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

Figure 1.

Figure 1

Inhibition of endosome-to-plasma membrane recycling restricts VEGFR2 to a perinuclear compartment. (A) Human umbilical vein endothelial cells (HUVECs) were pre-treated for 2 h with cycloheximide (CHX) prior to treatment with 20 μM monensin for zero, 30 or 120 min. Cells were then fixed, permeabilised and labelled with goat anti-VEGFR2 extracellular domain (green) and mouse anti-TfR (red). Primary antibodies were visualized using AlexaFluor-conjugated species-specific secondary antibodies, and the nucleus was labelled with DAPI (blue). Bar: 10 μm. Insets show a two-fold magnification of the indicated regions. (B) Serum-starved HUVECs were pre-treated with 20 μM monensin prior to VEGF-A stimulation for zero, 30 or 120 min. Cell lysates were immunoblotted with antibodies specific for VEGFR2 or actin. The data shown is representative of three independent experiments. (C) Flow cytometry analysis of HUVECs treated with either 20 μM monensin or 50 μg/mL cycloheximide (CHX) for zero, 30, 60 or 120 min. Error bars denote ± SEM (n = 3); *, p < 0.05.