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type=\u0022text\/css\u0022 rel=\u0022stylesheet\u0022 href=\u0022https:\/\/www.stormoverpacific.com\/sites\/default\/files\/advagg_css\/css__KwIbTmK5u_TU0NIDnj2cXC8qhcT3ubSigju5zweyvGo__wfUCN1ftcRJqvO1mXYEDxSMyoCswJBppiBXHDPwtaqM__MWGPudBpBmsaTs1FKLLZ0RHlWpbgNx0qxDcFgKr833o.css\u0022 media=\u0022all\u0022 \/\u003E\n\u003Clink rel=\u0027stylesheet\u0027 type=\u0027text\/css\u0027 href=\u0027\/sites\/all\/modules\/contrib\/panels\/plugins\/layouts\/onecol\/onecol.css\u0027 \/\u003E\u003C\/head\u003E\u003Cbody\u003E\u003Cdiv class=\u0022panels-ajax-tab-panel panels-ajax-tab-panel-jnl-template-cob-tab-data\u0022\u003E\u003Cdiv class=\u0022panel-display panel-1col clearfix\u0022 \u003E\n \u003Cdiv class=\u0022panel-panel panel-col\u0022\u003E\n \u003Cdiv\u003E\u003Cdiv class=\u0022panel-pane pane-cob-fragment-figures\u0022 \u003E\n \n \u003Ch2 class=\u0022pane-title\u0022\u003EArticle Figures \u0026amp; Tables\u003C\/h2\u003E\n \n \n \u003Cdiv class=\u0022pane-content\u0022\u003E\n \u003Cdiv class=\u0022elements-frag-data highwire-markup\u0022 id=\u0022fig-data\u0022\u003E\u003Cdiv id=\u0022fig-data-figures\u0022 class=\u0022group frag-figures\u0022\u003E\u003Cdiv class=\u0022fig-data-title-jump clearfix\u0022\u003E\u003Ch3 id=\u0022fig-frag-data-title\u0022 class=\u0022fig-data-group-title\u0022\u003EFigures\u003C\/h3\u003E\u003Cdiv class=\u0022fig-data-jump-links\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cdiv class=\u0022item-list\u0022\u003E\u003Cul id=\u0022fig-frag-fig\u0022 class=\u0022fig-frag-data-list clearfix\u0022\u003E\u003Cli class=\u0022first\u0022\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F1\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F1.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022\u0026#x3B1;-synuclein PMCA. (A) Workflow of \u0026#x3B1;-synuclein PMCA: lyophilized recombinant wild-type protein was reconstituted in buffer to 90\u0026#x2005;\u0026#xB5;M and 60\u0026#x2005;\u0026#xB5;l was aliquoted into PCR tubes. Samples were exposed to PMCA in the presence of beads. Sonications were set to occur for 20\u0026#x2005;s every 29\u0026#x2005;min 40\u0026#x2005;s for a total process time of 72\u0026#x2005;h. This figure was created with Biorender.com. (B\u0026#x2013;D) The extent of fibrillization in protein exposed to PMCA and non-PMCA controls (\u0026#x2212;80\u0026#xB0;C) was assessed by (B) western immunoblot analysis using \u0026#x3B1;-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118\u0026#x2013;123), (C) transmission electron microscopy (TEM) (representative images shown) and (D) thioflavin T (ThT) fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean\u0026#xB1;s.e.m. (n=3) and represent every experimental replicate performed.\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-955822383\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;\u0026#x3B1;-synuclein PMCA.\u0026lt;\/strong\u0026gt; (A) Workflow of \u0026#x3B1;-synuclein PMCA: lyophilized recombinant wild-type protein was reconstituted in buffer to 90\u0026#x2005;\u0026#xB5;M and 60\u0026#x2005;\u0026#xB5;l was aliquoted into PCR tubes. Samples were exposed to PMCA in the presence of beads. Sonications were set to occur for 20\u0026#x2005;s every 29\u0026#x2005;min 40\u0026#x2005;s for a total process time of 72\u0026#x2005;h. This figure was created with Biorender.com. (B\u0026#x2013;D) The extent of fibrillization in protein exposed to PMCA and non-PMCA controls (\u0026#x2212;80\u0026#xB0;C) was assessed by (B) western immunoblot analysis using \u0026#x3B1;-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118\u0026#x2013;123), (C) transmission electron microscopy (TEM) (representative images shown) and (D) thioflavin T (ThT) fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean\u0026#xB1;s.e.m. (\u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=3) and represent every experimental replicate performed.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 1.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F1.medium.gif\u0022 width=\u0022440\u0022 height=\u0022414\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 1.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F1.medium.gif\u0022 width=\u0022440\u0022 height=\u0022414\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F1.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 1.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F1.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339458\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 1.\u003C\/span\u003E \u003Cp id=\u0022p-10\u0022\u003E\u003Cstrong\u003E\u03b1-synuclein PMCA.\u003C\/strong\u003E (A) Workflow of \u03b1-synuclein PMCA: lyophilized recombinant wild-type protein was reconstituted in buffer to 90\u2005\u00b5M and 60\u2005\u00b5l was aliquoted into PCR tubes. Samples were exposed to PMCA in the presence of beads. Sonications were set to occur for 20\u2005s every 29\u2005min 40\u2005s for a total process time of 72\u2005h. This figure was created with \u003Ca href=\u0022http:\/\/Biorender.com\u0022\u003EBiorender.com\u003C\/a\u003E. (B\u2013D) The extent of fibrillization in protein exposed to PMCA and non-PMCA controls (\u221280\u00b0C) was assessed by (B) western immunoblot analysis using \u03b1-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118\u2013123), (C) transmission electron microscopy (TEM) (representative images shown) and (D) thioflavin T (ThT) fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean\u00b1s.e.m. (\u003Cem\u003En\u003C\/em\u003E=3) and represent every experimental replicate performed.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F2\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F2.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022Production of misfolded \u0026#x3B1;-synuclein species in PBSN buffer produces comparable misfolded content to \u0026#x3B1;-synuclein species generated in PCB. Lyophilized recombinant wild-type protein was reconstituted in PBSN (\u0026#x3B1;-synuclein:PBSN) or PCB (\u0026#x3B1;-synuclein:PCB) and subjected to PMCA for a total process time of 0, 24, 48 or 72\u0026#x2005;h. (A,B) Extent of fibrillization in samples was assessed using (A) western immunoblot analysis using \u0026#x3B1;-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118\u0026#x2013;123) and (B) silver staining, where the asterisk indicates saturation of the protein signal. (C) Analytical ultracentrifugation sedimentation velocity analyses of non-PMCA and PMCA-generated \u0026#x3B1;-synuclein. Continuous mass distribution [c(M)] distributions are shown as a function of molar mass. Inset shows zoomed-in image of values between 0 and 200\u0026#x2005;kDa. Best fits to experimental data yielded a root mean square deviation of 0.0055 and 0.0045, f\/f0 of 2.2 and 1.1, and Runs Test Z of 12 and 0.59 for non-PMCA and PMCA-generated \u0026#x3B1;-synuclein, respectively. (D) Secondary structure of non-PMCA (0\u0026#x2005;h) or PMCA-generated (24\u0026#x2005;h or 72\u0026#x2005;h) \u0026#x3B1;-synuclein:PBSN and \u0026#x3B1;-synuclein:PCB was determined using circular dichroism (CD) spectroscopy (n=1, representative spectra). (E) TEM of fibrils produced in PBSN after 72\u0026#x2005;h PMCA (representative image). (F) \u0026#x3B1;-Synuclein:PBSN exposed to PMCA for 72\u0026#x2005;h or non-PMCA (0\u0026#x2005;h) monomeric controls were further characterized by ThT fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean\u0026#xB1;s.e.m. (n=3) and represent every experimental replicate performed.\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-955822383\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;Production of misfolded \u0026#x3B1;-synuclein species in PBSN buffer produces comparable misfolded content to \u0026#x3B1;-synuclein species generated in PCB.\u0026lt;\/strong\u0026gt; Lyophilized recombinant wild-type protein was reconstituted in PBSN (\u0026#x3B1;-synuclein:PBSN) or PCB (\u0026#x3B1;-synuclein:PCB) and subjected to PMCA for a total process time of 0, 24, 48 or 72\u0026#x2005;h. (A,B) Extent of fibrillization in samples was assessed using (A) western immunoblot analysis using \u0026#x3B1;-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118\u0026#x2013;123) and (B) silver staining, where the asterisk indicates saturation of the protein signal. (C) Analytical ultracentrifugation sedimentation velocity analyses of non-PMCA and PMCA-generated \u0026#x3B1;-synuclein. Continuous mass distribution [\u0026lt;em\u0026gt;c\u0026lt;\/em\u0026gt;(\u0026lt;em\u0026gt;M\u0026lt;\/em\u0026gt;)] distributions are shown as a function of molar mass. Inset shows zoomed-in image of values between 0 and 200\u0026#x2005;kDa. Best fits to experimental data yielded a root mean square deviation of 0.0055 and 0.0045, \u0026lt;em\u0026gt;f\u0026lt;\/em\u0026gt;\/\u0026lt;em\u0026gt;f\u0026lt;\/em\u0026gt;\u0026lt;sub\u0026gt;0\u0026lt;\/sub\u0026gt; of 2.2 and 1.1, and Runs Test Z of 12 and 0.59 for non-PMCA and PMCA-generated \u0026#x3B1;-synuclein, respectively. (D) Secondary structure of non-PMCA (0\u0026#x2005;h) or PMCA-generated (24\u0026#x2005;h or 72\u0026#x2005;h) \u0026#x3B1;-synuclein:PBSN and \u0026#x3B1;-synuclein:PCB was determined using circular dichroism (CD) spectroscopy (\u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=1, representative spectra). (E) TEM of fibrils produced in PBSN after 72\u0026#x2005;h PMCA (representative image). (F) \u0026#x3B1;-Synuclein:PBSN exposed to PMCA for 72\u0026#x2005;h or non-PMCA (0\u0026#x2005;h) monomeric controls were further characterized by ThT fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean\u0026#xB1;s.e.m. (\u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=3) and represent every experimental replicate performed.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 2.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F2.medium.gif\u0022 width=\u0022335\u0022 height=\u0022440\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 2.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F2.medium.gif\u0022 width=\u0022335\u0022 height=\u0022440\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F2.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 2.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F2.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339453\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 2.\u003C\/span\u003E \u003Cp id=\u0022p-13\u0022\u003E\u003Cstrong\u003EProduction of misfolded \u03b1-synuclein species in PBSN buffer produces comparable misfolded content to \u03b1-synuclein species generated in PCB.\u003C\/strong\u003E Lyophilized recombinant wild-type protein was reconstituted in PBSN (\u03b1-synuclein:PBSN) or PCB (\u03b1-synuclein:PCB) and subjected to PMCA for a total process time of 0, 24, 48 or 72\u2005h. (A,B) Extent of fibrillization in samples was assessed using (A) western immunoblot analysis using \u03b1-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118\u2013123) and (B) silver staining, where the asterisk indicates saturation of the protein signal. (C) Analytical ultracentrifugation sedimentation velocity analyses of non-PMCA and PMCA-generated \u03b1-synuclein. Continuous mass distribution [\u003Cem\u003Ec\u003C\/em\u003E(\u003Cem\u003EM\u003C\/em\u003E)] distributions are shown as a function of molar mass. Inset shows zoomed-in image of values between 0 and 200\u2005kDa. Best fits to experimental data yielded a root mean square deviation of 0.0055 and 0.0045, \u003Cem\u003Ef\u003C\/em\u003E\/\u003Cem\u003Ef\u003C\/em\u003E\u003Csub\u003E0\u003C\/sub\u003E of 2.2 and 1.1, and Runs Test Z of 12 and 0.59 for non-PMCA and PMCA-generated \u03b1-synuclein, respectively. (D) Secondary structure of non-PMCA (0\u2005h) or PMCA-generated (24\u2005h or 72\u2005h) \u03b1-synuclein:PBSN and \u03b1-synuclein:PCB was determined using circular dichroism (CD) spectroscopy (\u003Cem\u003En\u003C\/em\u003E=1, representative spectra). (E) TEM of fibrils produced in PBSN after 72\u2005h PMCA (representative image). (F) \u03b1-Synuclein:PBSN exposed to PMCA for 72\u2005h or non-PMCA (0\u2005h) monomeric controls were further characterized by ThT fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean\u00b1s.e.m. (\u003Cem\u003En\u003C\/em\u003E=3) and represent every experimental replicate performed.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F3\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F3.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022PMCA-generated \u0026#x3B1;-synuclein selectively associates with cardiolipin. A hydrophobic membrane strip dotted with 15 different lipids (long-chain \u0026gt;diC16 synthetic analogues) was incubated with PMCA-generated \u0026#x3B1;-synuclein. (A) Binding of \u0026#x3B1;-synuclein to lipids was assessed via western immunoblotting using \u0026#x3B1;-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118\u0026#x2013;123). (B) Unlike PMCA-generated misfolded \u0026#x3B1;-synuclein, monomeric \u0026#x3B1;-synuclein (\u0026#x2212;80\u0026#xB0;C) did not show affinity to any lipid class, n=1. To determine whether cardiolipin (CA) modulates \u0026#x3B1;-synuclein fibrillization, CA was dissolved in MeOH\/PBSN (MeOH) and added to wild-type \u0026#x3B1;-synuclein to a final concentration of 5.7\u0026#x2005;\u0026#x3BC;M and 57\u0026#x2005;\u0026#xB5;M CA. MeOH\/PBSN alone added to \u0026#x3B1;-synuclein:PBSN served to control for the effect of the solvent. (C) Samples were subjected to PMCA for a total process time of 0, 6, 8, 10, 12, 24 and 72\u0026#x2005;h. Tubes containing PBSN only (no \u0026#x3B1;-synuclein) with solvent or CA exposed to PMCA for 0\u0026#x2005;h and 72\u0026#x2005;h served to detect any inherent fluorescence caused by CA or the solvent. Extent of fibrillization was assessed by measuring ThT fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean\u0026#xB1;s.e.m. (n=4 for all groups except for 0\u0026#x2005;h and 72\u0026#x2005;h, for which n=3) and represent every experimental replicate performed. To determine the effect of CA on \u0026#x3B1;-synuclein fibrillization compared to control samples, a mixed-effects ANOVA with Dunnett\u0027s multiple comparisons test was employed. *P\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-955822383\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;PMCA-generated \u0026#x3B1;-synuclein selectively associates with cardiolipin.\u0026lt;\/strong\u0026gt; A hydrophobic membrane strip dotted with 15 different lipids (long-chain \u0026gt;diC16 synthetic analogues) was incubated with PMCA-generated \u0026#x3B1;-synuclein. (A) Binding of \u0026#x3B1;-synuclein to lipids was assessed via western immunoblotting using \u0026#x3B1;-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118\u0026#x2013;123). (B) Unlike PMCA-generated misfolded \u0026#x3B1;-synuclein, monomeric \u0026#x3B1;-synuclein (\u0026#x2212;80\u0026#xB0;C) did not show affinity to any lipid class, \u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=1. To determine whether cardiolipin (CA) modulates \u0026#x3B1;-synuclein fibrillization, CA was dissolved in MeOH\/PBSN (MeOH) and added to wild-type \u0026#x3B1;-synuclein to a final concentration of 5.7\u0026#x2005;\u0026#x3BC;M and 57\u0026#x2005;\u0026#xB5;M CA. MeOH\/PBSN alone added to \u0026#x3B1;-synuclein:PBSN served to control for the effect of the solvent. (C) Samples were subjected to PMCA for a total process time of 0, 6, 8, 10, 12, 24 and 72\u0026#x2005;h. Tubes containing PBSN only (no \u0026#x3B1;-synuclein) with solvent or CA exposed to PMCA for 0\u0026#x2005;h and 72\u0026#x2005;h served to detect any inherent fluorescence caused by CA or the solvent. Extent of fibrillization was assessed by measuring ThT fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean\u0026#xB1;s.e.m. (\u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=4 for all groups except for 0\u0026#x2005;h and 72\u0026#x2005;h, for which \u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=3) and represent every experimental replicate performed. To determine the effect of CA on \u0026#x3B1;-synuclein fibrillization compared to control samples, a mixed-effects ANOVA with Dunnett\u0027s multiple comparisons test was employed. *\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;\u0026lt;0.05, **\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;\u0026lt;0.01. (D) ThT fluorescence was repeated in the presence of CA, MeOH or cholesterol (C), which showed no binding affinity to \u0026#x3B1;-synuclein in panel A. ThT was measured after 8\u0026#x2005;h, which represented the earliest detectable rise in ThT by CA observed in panel C. The values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. Data are presented as mean\u0026#xB1;s.e.m. (\u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=3) and represent every experimental replicate performed. (E) The biochemical properties of \u0026#x3B1;-synuclein species formed after 72\u0026#x2005;h PMCA (PMCA) or not (\u0026#x2212;80\u0026#xB0;C) was assessed by its resistance to proteolysis following digestion in proteinase K (PK) at a final concentration of 0, 2 or 10\u0026#x2005;\u0026#xB5;g\/ml. PK-resistant species were detected by immunoblotting using MJFR1. Areas of differential PK resistance between PMCA-generated species are identified by red arrows. \u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=1. (F) Ultracentrifugation was performed on \u0026#x3B1;-synuclein in the presence of 5.7\u0026#x2005;\u0026#xB5;M and 57\u0026#x2005;\u0026#xB5;M CA or buffer control to determine the insoluble load. \u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=1.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 3.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F3.medium.gif\u0022 width=\u0022348\u0022 height=\u0022440\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 3.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F3.medium.gif\u0022 width=\u0022348\u0022 height=\u0022440\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F3.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 3.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F3.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339455\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 3.\u003C\/span\u003E \u003Cp id=\u0022p-20\u0022\u003E\u003Cstrong\u003EPMCA-generated \u03b1-synuclein selectively associates with cardiolipin.\u003C\/strong\u003E A hydrophobic membrane strip dotted with 15 different lipids (long-chain \u0026gt;diC16 synthetic analogues) was incubated with PMCA-generated \u03b1-synuclein. (A) Binding of \u03b1-synuclein to lipids was assessed via western immunoblotting using \u03b1-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118\u2013123). (B) Unlike PMCA-generated misfolded \u03b1-synuclein, monomeric \u03b1-synuclein (\u221280\u00b0C) did not show affinity to any lipid class, \u003Cem\u003En\u003C\/em\u003E=1. To determine whether cardiolipin (CA) modulates \u03b1-synuclein fibrillization, CA was dissolved in MeOH\/PBSN (MeOH) and added to wild-type \u03b1-synuclein to a final concentration of 5.7\u2005\u03bcM and 57\u2005\u00b5M CA. MeOH\/PBSN alone added to \u03b1-synuclein:PBSN served to control for the effect of the solvent. (C) Samples were subjected to PMCA for a total process time of 0, 6, 8, 10, 12, 24 and 72\u2005h. Tubes containing PBSN only (no \u03b1-synuclein) with solvent or CA exposed to PMCA for 0\u2005h and 72\u2005h served to detect any inherent fluorescence caused by CA or the solvent. Extent of fibrillization was assessed by measuring ThT fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean\u00b1s.e.m. (\u003Cem\u003En\u003C\/em\u003E=4 for all groups except for 0\u2005h and 72\u2005h, for which \u003Cem\u003En\u003C\/em\u003E=3) and represent every experimental replicate performed. To determine the effect of CA on \u03b1-synuclein fibrillization compared to control samples, a mixed-effects ANOVA with Dunnett\u0027s multiple comparisons test was employed. *\u003Cem\u003EP\u003C\/em\u003E\u0026lt;0.05, **\u003Cem\u003EP\u003C\/em\u003E\u0026lt;0.01. (D) ThT fluorescence was repeated in the presence of CA, MeOH or cholesterol (C), which showed no binding affinity to \u03b1-synuclein in panel A. ThT was measured after 8\u2005h, which represented the earliest detectable rise in ThT by CA observed in panel C. The values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. Data are presented as mean\u00b1s.e.m. (\u003Cem\u003En\u003C\/em\u003E=3) and represent every experimental replicate performed. (E) The biochemical properties of \u03b1-synuclein species formed after 72\u2005h PMCA (PMCA) or not (\u221280\u00b0C) was assessed by its resistance to proteolysis following digestion in proteinase K (PK) at a final concentration of 0, 2 or 10\u2005\u00b5g\/ml. PK-resistant species were detected by immunoblotting using MJFR1. Areas of differential PK resistance between PMCA-generated species are identified by red arrows. \u003Cem\u003En\u003C\/em\u003E=1. (F) Ultracentrifugation was performed on \u03b1-synuclein in the presence of 5.7\u2005\u00b5M and 57\u2005\u00b5M CA or buffer control to determine the insoluble load. \u003Cem\u003En\u003C\/em\u003E=1.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F4\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F4.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022PMCA-generated misfolded \u0026#x3B1;-synuclein causes mitochondrial respiration to become hyperactive in SH-SY5Y cells. (A\u0026#x2013;F) SH-SY5Y cells were incubated with PMCA-generated \u0026#x3B1;-synuclein (PMCA), monomeric \u0026#x3B1;-synuclein (\u0026#x2212;80\u0026#xB0;C) or buffer alone or left untreated, which collectively represented the control group (Control). Medium-containing cells were plated into each of four wells per sample of a Seahorse XFe24 plate and mitochondria respiration was measured in adhered cells using the Seahorse XFe24 Analyzer. This was performed by detecting changes in oxygen (referred to as the oxygen consumption rate; OCR) following the addition of pharmacological agents: oligomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), rotenone and antimycin A. In doing so, the following parameters were measured: (A) basal OCR, (B) ATP synthesis, (C) max OCR, (D) complex I activity, (E) complex II activity and (F) non-mitochondrial respiration. The average of five wells was taken for each sample per experimental replicate. Data are presented as mean\u0026#xB1;s.e.m. (n=16, 13, 18 for PMCA, \u0026#x2212;80\u0026#xB0;C and Control, respectively) and represent every experimental replicate performed. Statistical significance was examined by ANOVA and Tukey\u0027s multiple comparisons test with a statistical criterion of 0.01. **P\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-955822383\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;PMCA-generated misfolded \u0026#x3B1;-synuclein causes mitochondrial respiration to become hyperactive in SH-SY5Y cells\u0026lt;em\u0026gt;.\u0026lt;\/em\u0026gt;\u0026lt;\/strong\u0026gt; (A\u0026#x2013;F) SH-SY5Y cells were incubated with PMCA-generated \u0026#x3B1;-synuclein (PMCA), monomeric \u0026#x3B1;-synuclein (\u0026#x2212;80\u0026#xB0;C) or buffer alone or left untreated, which collectively represented the control group (Control). Medium-containing cells were plated into each of four wells per sample of a Seahorse XFe24 plate and mitochondria respiration was measured in adhered cells using the Seahorse XFe24 Analyzer. This was performed by detecting changes in oxygen (referred to as the oxygen consumption rate; OCR) following the addition of pharmacological agents: oligomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), rotenone and antimycin A. In doing so, the following parameters were measured: (A) basal OCR, (B) ATP synthesis, (C) max OCR, (D) complex I activity, (E) complex II activity and (F) non-mitochondrial respiration. The average of five wells was taken for each sample per experimental replicate. Data are presented as mean\u0026#xB1;s.e.m. (\u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=16, 13, 18 for PMCA, \u0026#x2212;80\u0026#xB0;C and Control, respectively) and represent every experimental replicate performed. Statistical significance was examined by ANOVA and Tukey\u0027s multiple comparisons test with a statistical criterion of 0.01. **\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;\u0026lt;0.01, ***\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;\u0026lt;0.001, ****\u0026lt;em\u0026gt;P\u0026lt;\/em\u0026gt;\u0026lt;0.0001.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 4.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F4.medium.gif\u0022 width=\u0022355\u0022 height=\u0022440\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 4.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F4.medium.gif\u0022 width=\u0022355\u0022 height=\u0022440\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F4.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 4.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F4.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339450\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 4.\u003C\/span\u003E \u003Cp id=\u0022p-28\u0022\u003E\u003Cstrong\u003EPMCA-generated misfolded \u03b1-synuclein causes mitochondrial respiration to become hyperactive in SH-SY5Y cells\u003Cem\u003E.\u003C\/em\u003E\u003C\/strong\u003E (A\u2013F) SH-SY5Y cells were incubated with PMCA-generated \u03b1-synuclein (PMCA), monomeric \u03b1-synuclein (\u221280\u00b0C) or buffer alone or left untreated, which collectively represented the control group (Control). Medium-containing cells were plated into each of four wells per sample of a Seahorse XFe24 plate and mitochondria respiration was measured in adhered cells using the Seahorse XFe24 Analyzer. This was performed by detecting changes in oxygen (referred to as the oxygen consumption rate; OCR) following the addition of pharmacological agents: oligomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), rotenone and antimycin A. In doing so, the following parameters were measured: (A) basal OCR, (B) ATP synthesis, (C) max OCR, (D) complex I activity, (E) complex II activity and (F) non-mitochondrial respiration. The average of five wells was taken for each sample per experimental replicate. Data are presented as mean\u00b1s.e.m. (\u003Cem\u003En\u003C\/em\u003E=16, 13, 18 for PMCA, \u221280\u00b0C and Control, respectively) and represent every experimental replicate performed. Statistical significance was examined by ANOVA and Tukey\u0027s multiple comparisons test with a statistical criterion of 0.01. **\u003Cem\u003EP\u003C\/em\u003E\u0026lt;0.01, ***\u003Cem\u003EP\u003C\/em\u003E\u0026lt;0.001, ****\u003Cem\u003EP\u003C\/em\u003E\u0026lt;0.0001.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003Cli class=\u0022last\u0022\u003E\u003Cdiv class=\u0022element-fig-frag-data clearfix supplementary-material-caption\u0022\u003E\u003Cdiv class=\u0022highwire-markup\u0022\u003E\u003Cdiv xmlns=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022 id=\u0022content-block-markup\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cdiv class=\u0022fig-expansion \u0022 id=\u0022F5\u0022\u003E\u003Cspan class=\u0022highwire-journal-article-marker-start\u0022\u003E\u003C\/span\u003E\u003Cdiv class=\u0022highwire-figure\u0022\u003E\u003Cdiv class=\u0022fig-inline-img-wrapper\u0022\u003E\u003Cdiv class=\u0022fig-inline-img\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F5.large.jpg?width=800\u0026amp;height=600\u0026amp;carousel=1\u0022 title=\u0022PMCA-generated misfolded \u0026#x3B1;-synuclein does not cause functional deficits in mitochondria of SH-SY5Y cells. SH-SY5Y cells were incubated with PMCA-generated \u0026#x3B1;-synuclein (PMCA), monomeric \u0026#x3B1;-synuclein (\u0026#x2212;80\u0026#xB0;C) or either buffer alone or left untreated, which collectively represented the control group (Control). Medium-containing cells were plated into each of four wells per sample of a Seahorse XFe24 plate pre-coated with Matrigel. The Seahorse XFe24 Analyzer measured mitochondria respiration by detecting changes in oxygen (referred to as the OCR) following the addition of pharmacological agents: oligomycin, CCCP, rotenone and antimycin A. The functioning of mitochondria was determined as parameters expressed as % of max or basal OCR (depending on the functional readout). Spare capacity was measured as the difference between max and basal OCR, and proton leak was measured as the difference between basal and ATP synthesis and non-respiratory. The average of five wells was taken for each sample per experimental replicate. Data are presented as mean\u0026#xB1;s.e.m. (n=16, n=13, n=18 for groups PMCA, \u0026#x2212;80\u0026#xB0;C and Control, respectively) and represent every experimental replicate performed. Statistical significance was examined by ANOVA and Tukey\u0027s multiple comparisons test with a statistical criterion of 0.01. No statistical significance was found for all experimental comparisons.\u0022 class=\u0022highwire-fragment fragment-images colorbox-load\u0022 rel=\u0022gallery-fragment-images-955822383\u0022 data-figure-caption=\u0022\u0026lt;div class=\u0026quot;highwire-markup\u0026quot;\u0026gt;\u0026lt;div xmlns=\u0026quot;http:\/\/www.w3.org\/1999\/xhtml\u0026quot;\u0026gt;\u0026lt;strong\u0026gt;PMCA-generated misfolded \u0026#x3B1;-synuclein do\u0026lt;\/strong\u0026gt;\u0026lt;strong\u0026gt;es\u0026lt;\/strong\u0026gt; \u0026lt;strong\u0026gt;not cause functional deficits in mitochondria of SH-SY5Y cells.\u0026lt;\/strong\u0026gt; SH-SY5Y cells were incubated with PMCA-generated \u0026#x3B1;-synuclein (PMCA), monomeric \u0026#x3B1;-synuclein (\u0026#x2212;80\u0026#xB0;C) or either buffer alone or left untreated, which collectively represented the control group (Control). Medium-containing cells were plated into each of four wells per sample of a Seahorse XFe24 plate pre-coated with Matrigel. The Seahorse XFe24 Analyzer measured mitochondria respiration by detecting changes in oxygen (referred to as the OCR) following the addition of pharmacological agents: oligomycin, CCCP, rotenone and antimycin A. The functioning of mitochondria was determined as parameters expressed as % of max or basal OCR (depending on the functional readout). Spare capacity was measured as the difference between max and basal OCR, and proton leak was measured as the difference between basal and ATP synthesis and non-respiratory. The average of five wells was taken for each sample per experimental replicate. Data are presented as mean\u0026#xB1;s.e.m. (\u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=16, \u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=13, \u0026lt;em\u0026gt;n\u0026lt;\/em\u0026gt;=18 for groups PMCA, \u0026#x2212;80\u0026#xB0;C and Control, respectively) and represent every experimental replicate performed. Statistical significance was examined by ANOVA and Tukey\u0027s multiple comparisons test with a statistical criterion of 0.01. No statistical significance was found for all experimental comparisons.\u0026lt;\/div\u0026gt;\u0026lt;\/div\u0026gt;\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cspan class=\u0022hw-responsive-img\u0022\u003E\u003Cimg class=\u0022highwire-fragment fragment-image lazyload\u0022 alt=\u0022Fig. 5.\u0022 src=\u0022data:image\/gif;base64,R0lGODlhAQABAIAAAAAAAP\/\/\/yH5BAEAAAAALAAAAAABAAEAAAIBRAA7\u0022 data-src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F5.medium.gif\u0022 width=\u0022366\u0022 height=\u0022440\u0022\/\u003E\u003Cnoscript\u003E\u003Cimg class=\u0022highwire-fragment fragment-image\u0022 alt=\u0022Fig. 5.\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F5.medium.gif\u0022 width=\u0022366\u0022 height=\u0022440\u0022\/\u003E\u003C\/noscript\u003E\u003C\/span\u003E\u003C\/a\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cul class=\u0022highwire-figure-links inline\u0022\u003E\u003Cli class=\u0022download-fig first\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F5.large.jpg?download=true\u0022 class=\u0022highwire-figure-link highwire-figure-link-download\u0022 title=\u0022Download Fig. 5.\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload figure\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022new-tab\u0022\u003E\u003Ca href=\u0022https:\/\/www.stormoverpacific.com\/content\/dmm\/13\/1\/dmm040899\/F5.large.jpg\u0022 class=\u0022highwire-figure-link highwire-figure-link-newtab\u0022 target=\u0022_blank\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EOpen in new tab\u003C\/a\u003E\u003C\/li\u003E\u003Cli class=\u0022download-ppt last\u0022\u003E\u003Ca href=\u0022\/highwire\/powerpoint\/1339449\u0022 class=\u0022highwire-figure-link highwire-figure-link-ppt\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003EDownload powerpoint\u003C\/a\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003Cdiv class=\u0022fig-caption\u0022 xmlns:xhtml=\u0022http:\/\/www.w3.org\/1999\/xhtml\u0022\u003E\u003Cspan class=\u0022fig-label\u0022\u003EFig. 5.\u003C\/span\u003E \u003Cp id=\u0022p-30\u0022\u003E\u003Cstrong\u003EPMCA-generated misfolded \u03b1-synuclein do\u003C\/strong\u003E\u003Cstrong\u003Ees\u003C\/strong\u003E \u003Cstrong\u003Enot cause functional deficits in mitochondria of SH-SY5Y cells.\u003C\/strong\u003E SH-SY5Y cells were incubated with PMCA-generated \u03b1-synuclein (PMCA), monomeric \u03b1-synuclein (\u221280\u00b0C) or either buffer alone or left untreated, which collectively represented the control group (Control). Medium-containing cells were plated into each of four wells per sample of a Seahorse XFe24 plate pre-coated with Matrigel. The Seahorse XFe24 Analyzer measured mitochondria respiration by detecting changes in oxygen (referred to as the OCR) following the addition of pharmacological agents: oligomycin, CCCP, rotenone and antimycin A. The functioning of mitochondria was determined as parameters expressed as % of max or basal OCR (depending on the functional readout). Spare capacity was measured as the difference between max and basal OCR, and proton leak was measured as the difference between basal and ATP synthesis and non-respiratory. The average of five wells was taken for each sample per experimental replicate. Data are presented as mean\u00b1s.e.m. (\u003Cem\u003En\u003C\/em\u003E=16, \u003Cem\u003En\u003C\/em\u003E=13, \u003Cem\u003En\u003C\/em\u003E=18 for groups PMCA, \u221280\u00b0C and Control, respectively) and represent every experimental replicate performed. Statistical significance was examined by ANOVA and Tukey\u0027s multiple comparisons test with a statistical criterion of 0.01. No statistical significance was found for all experimental comparisons.\u003C\/p\u003E\u003Cdiv class=\u0022sb-div caption-clear\u0022\u003E\u003C\/div\u003E\u003C\/div\u003E\u003Cspan class=\u0022highwire-journal-article-marker-end\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003Cspan id=\u0022related-urls\u0022\u003E\u003C\/span\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/li\u003E\u003C\/ul\u003E\u003C\/div\u003E\u003C\/div\u003E\u003C\/div\u003E \u003C\/div\u003E\n\n \n \u003C\/div\u003E\n\u003Cdiv class=\u0022panel-separator\u0022\u003E\u003C\/div\u003E\u003Cdiv class=\u0022panel-pane pane-earthchem\u0022 \u003E\n \n \n \n \u003Cdiv class=\u0022pane-content\u0022\u003E\n \u003Ca href=\u0022http:\/\/ecp.iedadata.org\/doidata\/10.1242\/dmm.040899\u0022 class=\u0022\u0022 data-icon-position=\u0022\u0022 data-hide-link-title=\u00220\u0022\u003E\u003Cimg src=\u0022http:\/\/ecp.iedadata.org\/doibanner\/10.1242\/dmm.040899\u0022 alt=\u0022\u0022 \/\u003E\u003C\/a\u003E \u003C\/div\u003E\n\n \n \u003C\/div\u003E\n\u003C\/div\u003E\n \u003C\/div\u003E\n\u003C\/div\u003E\n\u003C\/div\u003E\u003Cscript type=\u0022text\/javascript\u0022 src=\u0022https:\/\/www.stormoverpacific.com\/sites\/default\/files\/js\/js_hZg96SP9gBcOluDp2mGc57d8sP8uJ7g8P_JYsCISOgQ.js\u0022\u003E\u003C\/script\u003E\n\u003C\/body\u003E\u003C\/html\u003E"}Ļ