Figure 5.

Rab25 Enhances EGFR Recycle and Increases Cell Surface Content of EGFR

(A) Immunoblotting of EGFR on cell surface in A549 and A549R cells. PMCA1 was used as plasma membrane loading control, and Hsp90 was used as cytosol protein control.

(B–D) (B) The kinetics of ligand-induced internalization of EGFR in radioresistant CNE2R cell or A549R cell. Phosphorylated tyrosine (p-Tyr) was immunoprecipitated from CNE2R cells with knockdown (C) or overexpression (D) of Rab25, followed with immunoblotting of EGFR.

(E) Endocytotic rate of EGF in CNE2R cell or CNE2R cells with down-expression of Rab25. The amount of alex-488 conjugated EGF was quantified by flow cytometry.

(F) Quantification of the cell surface content of EGFR in cells or in cells with down-expression of Rab25. EGFR cell surface content was performed every 5 min after stimulation with 1 ng/mL EGF. Right, flow cytometry analysis at each time point was plotted as histogram.

(G) Left, cell surface EGFR recycling rate in H358 or H358R cells pretreated with 1 ng/mL EGF for 10 min before which cells had been starved for 6 h. Right: flow cytometry analysis at each time point was plotted as histogram.

(H and I) (H) Cell surface EGFR recycling rate in CNE2 and CNE2 cells stably transfected with Rab25 or in ovarian carcinoma taxol-resistant SKOV3R cells with knockdown of Rab25 (I). Mean ± SD, ∗∗p < 0.01.

(J) EGF-induced activation of RTK-mediated signaling pathways in A549R cells and A549R with siRNA-mediated knockdown of Rab25. Cells were first incubated for 6 h in serum-free medium and then treated with 1 ng/mL EGF for indicated time.

(K) Immunoblotting of radiation-induced activation of RTK pathways in A549R cells or in A549R with siRNA-mediated knockdown of Rab25. Cells were irradiated at 4 Gy.