Fig. 4. TCPTP dephosphorylates VEGFR2 in a phosphosite-specific manner and controls its activity. (A,B) HEK293 cells were transfected with VEGFR2, treated with VEGF and immunoprecipitated (IP) with anti-VEGFR2 antibody. Immunoprecipitates were incubated in the presence of recombinant TCPTP (TC45) or buffer control, and immunoblotted using total phosphotyrosine antibody (A) or phospho-specific VEGFR2 antibodies (B). Total VEGFR2 was blotted as a control. Representative blots out of three experiments with similar results are shown. The values under the blots represent VEGFR2 phosphorylation relative to the buffer controls (mean ± s.e.m.; n=3). (C,D) TCPTP activity controls the internalization of VEGFR2 in endothelial cells. (C) Subconfluent HUVECs were transfected with vector control (pCG) or constitutively active TCPTP (TC37), stimulated (+) or not (–) with VEGF, and stained for VEGFR2 (red) and nuclei (blue) after fixation and permeabilization. Arrows indicate representative VEGFR2 vesicles. (C, bottom) The number of VEGFR2-positive vesicles (mean ± s.e.m.; ^***P<0.005) was quantitated using image analysis. The perinuclear bright Golgi-resembling staining was excluded in the analysis. Scale bar: 10 µm. (D) HUVECs were transfected with TC37, TC45 or vector (pCG), or left untransfected, and surface-labelled with cleavable biotin. VEGFR2 was allowed to internalize for 15 minutes in the presence or absence of VEGF (100 ng/ml). Biotin remaining on the cell surface was cleaved and VEGFR2 was immunoprecipitated using anti-VEGFR2 antibody. Internalized VEGFR2 was detected by blotting for biotin. Because biotin is cleavable by reducing reagents, a non-reducing gel was used, and this alters the mobility of the proteins compared to a reducing gel. The numbers below the blot show levels of internalized VEGFR2 (mean ± s.d., two individual experiments).