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. 2014 Apr 29;3(2):363–385. doi: 10.3390/cells3020363

Figure 3.

Figure 3

Wild-type and mutant Rab4a-S22N blocks VEGF-A-stimulated VEGFR2 degradation. (A–C) HUVECs were transfected with GFP-Rab4a (green) and cells were then stimulated with VEGF-A for (A) 0 min, (B) 30 min or (C) 120 min in the presence of CHX. Cells were subsequently fixed, permeabilised and labelled with goat anti-VEGFR2 followed by AlexaFluor-conjugated secondary antibody (red). The nucleus is visualized using DAPI (blue). Inset panels show a two-fold magnification of boxed highlighted regions. Bar: 10 μm. (D–F) HUVECs were transiently transfected to express dominant-negative GDP-bound GFP-Rab4a-S22N (green) and then stimulated with VEGF-A for (D) 0 min, € 30 min or (F) 120 min in the presence of cycloheximide (CHX) and processed for immunofluorescence microscopy. VEGFR2 was detected using goat anti-VEGFR2 followed by AlexaFluor-conjugated secondary antibody (red), whilst the nuclear DNA was labelled with DAPI (blue). The images shown are representative of three independent experiments. Inset panels show a two-fold magnification of boxed highlighted regions. Bar: 10 μm.