Fig 1: C99's cholesterol-binding domain is necessary for cholesterol trafficking to MAM AScheme of PhotoClick cholesterol methodology to detect C99 interaction with cholesterol.BRepresentative immunoblot to reveal C99 levels in total homogenate (TH) and MAM fractions of APP-DKO cells expressing C99WT or C99MUT before (input) and after cholesterol pull-down. Acsl4 was used as a MAM marker. Streptavidin-HRP was used to detect total biotinylation (biotin conjugated to PhotoClick cholesterol).CQuantification of pulled-down C99 levels vs. input from the experiment in (B). Two-way repeated measures ANOVA (Fraction, Mutation) (n = 5; *P < 0.05, **P < 0.01).DImmunoblot showing the levels of pulled-down PhotoClick Cholesterol (streptavidin-HRP) in ER and MAM fractions. Note how the levels of pulled-down C99 in the ER are negligible when compared to those from MAM. Isolated MAM and ER fractions were assessed by Western blot (shown in Appendix Fig S1E).EQuantification of free cholesterol levels (FC) analyzed by lipidomics after subcellular fractionation to obtain MAM from the indicated cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol%). Graphs represent percentage over controls. Dashed line represents control levels. One-way ANOVA (n = 4; *P < 0.05).F, GQuantification of cholesterol uptake and esterification in the indicated cells was measured by 4 h incubation with 3H-cholesterol and subsequent analysis of radiolabel incorporation. The dashed line indicates control levels. Graphs represent fold change over controls. Treatment with Sandoz 58-035, a specific ACAT1 inhibitor, caused a ˜95% reduction in cholesterol esterification. EV, empty vector. One-sample t-test (n = 3–4; *P < 0.05).HRatio of cholesteryl esters:free cholesterol (CE:FC) in the indicated cells. One-way ANOVA (n = 4; *P < 0.05). Source data are available online for this figure.
Fig 2: The cholesterol-binding domain of C99 is necessary for its localization to MAM ACrude membrane fractions from APP-DKO cells expressing C99WT or C99MUT were treated with 0.2% Triton X-100, loaded onto continuous density sucrose gradients and centrifuged for 16 h. Fractions from these gradients were analyzed by Western blot to determine the migration of the indicated proteins [two parallel gels (bold vertical line)].B–GGraphs represent the relative abundance of the indicated proteins in each fraction of the gradient, as measured by densitometry analysis of Western blot signals (ImageJ). Note the reduced degree of comigration of C99MUT with MAM markers (Acsl4), compared to C99WT. Source data are available online for this figure.
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