Fig. 4
RAB37 knockdown interferes with formation of ATG12-ATG5-ATG16L1 complex. a ATG5 interacts with RAB37 and ATG16L1 respectively. Two cell lines (RAB37 knockdown and control HeLa cells) were cultured and then starved for 1 h before harvest. The lysates were immunoprecipitated with anti-ATG5 antibody or negative serum, followed by immunoblotting with the anti-RAB37, anti-ATG16L1 or anti-ATG5 antibody. b RAB37 knockdown interferes with co-localization of endogenous ATG5 and ATG16L1 proteins. RAB37 overexpression, RAB37 knockdown and wild type HeLa cells were starved in the medium EBSS for 1 h before stained by immuno fluorescence with anti-ATG5, anti-ATG16L1 antibodies and then anti-mouse IgG FITC conjugated antibody (green), or anti-rabbit IgG TRITC conjugated antibody (red) respectively. The nuclei were counterstained with DAPI (blue). Confocal images were taken. Scale bar, 10 µm. Percentages of co-localized dots (ATG5 + ATG16L1 (yellow)/ATG5 (green)) in three HeLa cell lines were determined and the co-localized dots between ATG5 and ATG16L1 were counted from ~15 cells respectively. * stands for P < 0.05. c RAB37 knockdown affects formation of ATG12-ATG5-ATG16L1 complex. Lysates of three cell lines (RAB37 overexpression, RAB37 knockdown and wild type HeLa cells) were separated using continuous sucrose gradients (0–30%). Fractions were subjected to Western blotting using the indicated antibodies. Red triangles indicate the size shift of the complex
