Fig. 3: Arl4A affects the level of EGFR ubiquitination and sorting of EGFR into MVBs.

a HeLa cells were transfected with empty vector (EV) or Arl4A WT in combination with EGFR-Myc (b) C33-A cells were transfected with either control or Arl4A siRNA in combination with EGFR-Myc. a and b After 24 h of transfection with plasmids or 48 h of transfection with siRNAs, the cells were starved and stimulated with EGF (100 ng/ml) for the indicated time. Cell lysates were immunoprecipitated with Myc-Trap for EGFR-Myc and analyzed by Western blotting with antibodies against ubiquitin or Myc. Total cell lysates (input) were immunoblotted with the indicated antibodies. The amounts of ubiquitin-EGFR were determined by ImageJ software. The results represent the mean ± SD of three independent experiments and the p-values were assessed by a two-sided t-test. c HeLa cells were transfected with either empty vector (EV) or Arl4A and GFP-Rab5A Q79L for 24 h. After serum starvation, the cells were incubated with 100 ng/ml EGF for 10 min at 37 °C and then stained with anti-EGFR (red) and anti-Arl4A (blue) antibodies. Images were acquired using a Leica TCS SP8 STED microscope. Scale bar, 5 µm. Imaris 3D visualization software was used to create an enlarged endosome in a 3D orthoslicer view. Endosomes with diameters >2 μm were identified for analysis. Using the Rab5-Q79L outline to define the ROI, the EGFR intensities in the Rab5-Q79L-positive MVB lumen were measured. The results represent the mean ± SD of three independent experiments, each consisting of 26 or more endosome scans from ~10 cells each consisting of 26 or more endosome scans from ~10 cells (total numbers: EV = total 100 endosomes from 30 cells; Arl4A = total 81 endosomes from 30 cells; p-value was assessed by two-sided t-test). Source data are provided as a Source Data file.