Fig. 7: Arl4A binding modulates the association of VPS36 with MVBs.

a HeLa cells were transfected for 24 h with the indicated plasmid. Confocal sections of HeLa cells stained with anti-Myc (VPS36-WT-Myc or VPS36-A6-Myc) (green), anti-CD63 (red), and DAPI (blue). Scale bar, 10 µm. Quantification of VPS36-Myc and CD63 colocalization is shown as the Pearson coefficient. The results represent the mean ± SD of three independent experiments and the p-value was assessed by a two-sided t-test (VPS36-WT = 84 cells, VPS36-A6 = 104 cells). b HeLa cells were co-transfected with VPS36-WT-Myc or VPS36-A6-Myc and GFP-Rab5A Q79L for 24 h. The cells were fixed and then stained with anti-Myc (red) and anti-GFP (green) antibodies. The images were acquired using the Leica TCS SP8 STED microscope. Scale bar, 5 µm. Imaris software was used to quantify the 3D-colocalization coefficient values of VPS36 and Rab5 Q79L. The results represent the mean ± SD of three independent experiments and the p-value was assessed by a two-sided t-test (VPS36-WT = 16 cells, VPS36-A6 = 22 cells). c HeLa cells stably expressing control shRNA or VPS4A and VPS4B shRNA using shRNA–expressing lentivirus. After puromycin selection, the cells were transfected with the indicated plasmids. Confocal sections of HeLa cells stained with anti-Myc (VPS36-WT-Myc or VPS36-A6-Myc) (green), anti-CD63 (red), and DAPI (blue). Scale bar, 10 µm. Quantification of colocalization of VPS36-Myc and CD63 presented as colocalization coefficient using the software ZEN. Results represent the mean ± SD of three independent experiments, and the p values were determined by one-way ANOVA with Tukey’s test (shControl/VPS36- WT = 40 cells, shVPS36/VPS36- WT = 41 cells, shControl/VPS36-A6 = 52 cells, shVPS36/VPS36-A6 = 42 cells). Source data are provided as a Source Data file.