Fig. 1: Alr4A depletion promotes EGFR lysosomal degradation.
From: Endosomal Arl4A attenuates EGFR degradation by binding to the ESCRT-II component VPS36
a HeLa cells transfected with the indicated siRNAs for 48 h were serum-starved and treated with EGF-Alexa Fluor 555 at a concentration of 80 ng/mL for the indicated times. Scale bar, 10 µm. b The EGF-Alexa-Fluor 555 intensity was determined using a nonlinear one-phase decay fit of the time course. The results represent the mean fluorescence intensity ± SD of three independent experiments and the p-values were assessed by a two-sided t-test. Arl4A-knockdown efficiency was assessed by RT-PCR. c C33-A cells were treated with the indicated siRNAs. The results represent the mean ± SD of three independent experiments and the p-values were assessed by a two-sided t-test. d The effects of Arl4A knockdown on EGFR protein levels were rescued by overexpression of Arl4ARes. C33-A cells were treated with either the control or Arl4A siRNA as well as the indicated plasmids. Quantification of EGFR and c-Met levels is shown in the right panel. The results represent the mean ± SD of three independent experiments and the p-values were assessed by one-way ANOVA with Tukey’s test. e C33-A cells were treated with either the control or Arl4A siRNA and incubated with NH4Cl (10 mM) or MG132 (10 mM) for 6 h. Quantification of EGFR levels is shown in the lower panel. The results represent the mean ± SD of four independent experiments and the p-values were assessed by one-way ANOVA with Tukey’s test. f and g Arl4A knockdown and expression regulated the half-life of EGFR degradation upon EGF treatment. Arl4A was knocked down in C33A cells (f) and overexpressed in HeLa cells (g) with the indicated siRNAs and plasmids. The results represent the mean ± SD of three independent experiments. To compare the EGFR degradation rates, we adjusted the EGFR protein levels at time 0 in each group to be the same for western blot analysis. The t 1/2 ± SD of EGFR was obtained using a nonlinear one-phase decay fit of the time course; p-values were assessed by a two-sided t-test. Source data are provided as a Source Data file.
