Journal:

Article Title: Src-mediated Phosphorylation of Hsp90 in Response to Vascular Endothelial Growth Factor (VEGF) Is Required for VEGF Receptor-2 Signaling to Endothelial NO Synthase

doi: 10.1091/mbc.E07-05-0467

Figure Lengend Snippet: Dynamin-dependent internalization of VEGFR-2. (A) K44A-dynamin inhibits VEGFR-2 surface expression. HUVECs infected with adenoviruses coding for β-galactosidase (Ad-β-gal) or HA-K44A-dynamin (Ad-HA-K44A-dyn) were serum-starved and stimulated with VEGF (40 ng/ml, 30 min). The cell surface expression of VEGFR-2 was analyzed by FACS. Inset, cells were lysed, and HA-K44A-dyn expression was monitored by Western blot (wb) against HA. (B) Inhibition of VEGF-stimulated subcellular relocalization of VEGFR-2 by K44A-dynamin. BAECs were transfected with VEGFR-2-GFP and HA-tagged WT- or K44A-dynamin. Transfected BAECs were stimulated or not with VEGF (40 ng/ml; 30 min). Cell were fixed permeabilized, and immunofluorescence was performed using anti-GFP (green) and anti-HA (red) antibodies. Transfected BAECs, expressing both proteins (merged image), were visualized using a confocal microscope. White arrows point to membrane localized VEGFR-2-GFP. Note that the nuclear anti-GFP staining is nonspecific (not shown).

Article Snippet: VEGFR-2-green fluorescent protein (GFP) was detected using rabbit anti-GFP antibody (Cedarlane Laboratories, Hornby, ON, Canada).

Techniques: Expressing, Infection, Western Blot, Inhibition, Transfection, Immunofluorescence, Microscopy, Staining