Figure 2.

RPTPβ/ζ is required for VEGF ₁₆₅ -induced cell surface NCL localization. Serum starved HUVEC were treated with VEGF₁₆₅ (10 ng/ml) for 10 min. Cell lysates were analyzed by Western blot for non Tyr530 phosphorylated (npc-Src), Tyr419 phosphorylated (pc-Src) and total (tc-Src) c-Src (A), as well as for phospho-β₃Y773 (pβ₃Y773) and total β₃ (tβ₃) integrin (B). Numbers in brackets denote the average-fold change of the ratio npc-Src:tc-Src, pc-Src:tc-Src or pβ₃Y773:tβ₃ respectively, compared with the corresponding non stimulated, untransfected cells (set as default 1). (C) Representative immunofluorescence images stained for NCL (green), RPTPβ/ζ (red) and nucleus (blue) from serum starved HUVEC treated with VEGF₁₆₅ (10 ng/ml) for 5 h at 37°C. (D) Representative immunofluorescence images stained for NCL (green) and nucleus (blue) from serum starved VEGF₁₆₅-stimulated HUVEC in the presence or absence of inhibitors for c-Src (PP1 10 μΜ), PI3K (wortmannin 100 nM) and ERK½ (U0126 20 nM). Scale bars in C and D correspond to 10 μm. (E) Lysates from serum starved VEGF₁₆₅-stimulated HUVEC in the presence or absence of PP1 and wortmannin, were analyzed by Western blot for pβ₃Y773 and tβ₃ integrin. Numbers in brackets denote the average-fold change of the ratio pβ₃Y773:tβ₃ compared with untreated cells (set as default 1). (F and G) Phosphorylation of PI3K in HUVEC and CHO cells respectively. Data are expressed as mean ± s.e.m percentage change in PI3K compared with the untreated cells (set as default 100). In all cases, data come from three independent experiments. siNeg, HUVEC transfected with a negative control siRNA; siRPTPβ/ζ1, HUVEC transfected with siRPTPβ/ζ#1; siRPTPβ/ζ2, HUVEC transfected with si RPTPβ/ζ#2; vector, CHO cells transfected with the plasmid vector; wtβ₃, CHO cells over-expressing wild-type β₃; β₃Y773F, CHO cells over-expressing β₃Y773F; β₃Y785F, CHO cells over-expressing β₃Y785F.