FIGURE 6:

Rab20 knockdown accelerates endocytic trafficking and degradation in macrophages. (A) Colocalization of EGF with endogenous Rab20 (left) or overexpressed Rab20 (right) after 10 min of internalization. Insets show regions of interest indicated by the white rectangles. Scale bar, 10 μm. (B) Quantification of internalized EGF at 10 min of incubation. For each group, at least 50 cells were analyzed. Data show mean ± SEM of one representative experiment from three independent experiments; ns: nonsignificant. (C) EGF-stimulated endocytosis and postendocytic trafficking in macrophages expressing scrambled shRNA or Rab20 shRNA. Alexa Fluor 488–conjugated EGF was added to macrophages for 1 h on ice and incubated for the indicated time at 37°C. After fixation, cells were stained for LAMP-2. Insets show regions of interest indicated by the white rectangles. Scale bar, 10 μm. (D) Quantification of the colocalization of Alexa Fluor 488–conjugated EGF and LAMP-2. For each group, at least 50 cells were analyzed. Data show mean ± SEM of one representative experiment from three independent experiments. The p values were calculated using Student's two-tailed t test. *p ≤ 0.05. (E) Western blot analysis of EGFR levels upon EGF stimulation in macrophages expressing scrambled shRNA or Rab20 shRNA. RAW264.7 macrophages stably expressing scrambled shRNA or Rab20 shRNA were incubated with EGF for 1 h on ice and incubated for the indicated time at 37°C. Cells were collected, and cell lysates were blotted with anti-EGFR antibody. Actin was used as loading control. (F) Quantification of EGFR levels in macrophages expressing scrambled shRNA or Rab20 shRNA at different time points. Relative intensity of EGFR was calculated considering the loading control. Data show mean ± SEM of two representative experiments.