Figure 6.

Analysis of RAB4-dependent VEGFR2 trafficking. A, assessment of surface VEGFR2 levels. HUVEC transfected with scrambled siRNA (control), scrambled siRNA and siRNA targeting RABEPS (siRABEP2), scrambled siRNA and siRNA targeting RAB4a and RAB4b (siRAB4), or siRNA targeting Rab4a, Rab4b, and RABEP2 (siRab4/siRABEP2). HUVEC were serum starved for 12 h, incubated with biotin to label surface proteins, and biotin-labeled proteins were probed for VEGFR2 (surface VEGFR2). 5% input was loaded and probed for VEGFR2 (total VEGFR2), RABEP2, and Rab4 to validate knockdown. B, HUVEC transfected with scrambled siRNA (control) and siRNA targeting RAB4a and RAB4b transcripts (siRAB4) were serum starved for 12 h and then stimulated with 50 ng/ml of VEGF-A₁₆₅ for 0, 5, or 15 min. Lysates were collected and probed using antibodies against the tyrosine 1175 phosphorylation site of VEGFR2 (pVEGFR2 Y1175), total VEGFR2, the serine 473 phosphorylation site of AKT (pAKT S473), total AKT, phosphorylated ERK1 and -2 (pERK1/2), total ERK1 and -2 (ERK1/2), RAB4 to validate knockdown, and GAPDH as a loading control. C, quantification of pVEGFR2 (Tyr¹¹⁷⁵) normalized to total VEGFR2 levels. D, quantification of pAkt (Ser⁴⁷³) normalized to total Akt. E, quantification of pERK1/2 normalized to total ERK1/2. C–E, quantification based on 4 independent experiments (mean ± S.D., NS, not significant; *, p < 0.05).