Fig. 2. Phosphorylation of Rab7a on S72 promotes late EGFR endocytosis.

A Cluster analysis of differentially expressed proteins and phosphopeptides in RAW264.7 measured after treated with LPS (1 µg/mL) for 30 min with or without PD168393 (PD 10 µM) pretreatment for 30 min, when compared with control. Blue and red indicates down-or upregulation, respectively (n = 3 samples of each condition). B PD168393 (PD 10 µM) inhibited Rab7a phosphorylation in response to LPS (30 min). The indicated cells were subjected to depolarization, and cell extracts were subjected to phostag-PAGE with Rab7a antibody by immunoblotting. C Stable expression of Rab7a-WT, Rab7a-S72A or Rab7a-S72E mutant in Rab7a shRNA knockdown RAW264.7 cells. Rab7a total protein expression was measure by western blot. a-Tubulin as a loading control. D a-Rab7a immunoprecipitates from the indicated cells were separated by phostag-PAGE followed by immunoblotting with a-Rab7a antibodies. E Immune-staining of Flag-Rab7a and CD63 in the indicated Rab7a mutant RAW264.7 cells followed by LPS treatment (1 µg/mL) for 30 min. Scale bar, 5 μm. F Immune-staining of EGFR and LAMP1 in the indicated Rab7a mutant RAW264.7 cells followed by LPS treatment (1 µg/mL) for 24 h. Scale bar, 5 μm. G–I The indicated Rab7a mutant RAW264.7 cells followed by LPS treatment (1 µg/mL) for 24 h. G EGFR expression on the surface of RAW264.7 was analyzed by flow cytometry. H Percentage of EGFR-positive RAW264.7 is shown (n = 3). I Mean fluorescence intensity (MFI) is shown (n = 3). J Cell lysates from the indicated Rab7a mutant RAW264.7 cells followed by LPS treatment (1 µg/mL) for 24 h were prepared, and phospho-ERK, total ERK, total p38 and phospho-p38 protein expression was measure by western blot. The graphs depict mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.