Fig. 3. FAK, RhoA and pp60^src activation by Tat in ECs. (A) GM7373 cells were allowed to adhere to plastic coated with 20 µg/ml of GST-Tat or PL, or were maintained in suspension (s) for 30 minutes. Then, cells were lysed, and analysed for FAK, RhoA or pp60^src activation (a, activated/phosphorylated protein; t, total protein). (B,C) GM7373 cells adherent to cell culture plates were incubated for 30 minutes at 37°C with the indicated concentrations of free GST-Tat (B) or with 100 ng/ml of GST-Tat for the indicated periods of time (C). Then, cells were lysed, immunoprecipitated with anti-FAK antibodies and immunoblotted with anti-phosphotyrosine antibody. (D) GM7373 cells adherent to cell culture plates were incubated for 30 minutes at 37°C with 100 ng/ml of GST-Tat, lysed, and immunoblotted with antibodies specific for the indicated FAK phosphotyrosine residues. (E) GM7373 cells adherent to cell culture plates were incubated for 30 minutes at 37°C with or without GST-Tat (100 ng/ml) in the absence or in the presence of anti-_vß₃ (LM 609) or irrelevant (N.I.) antibodies (both at 75 µg/ml) or of cRGDfV and cRADfV peptides (both at 3 µM). Then, cells were lysed and immunoblotted with antibodies specific for FAK phospho-Tyr₃₉₇ residue. In B, C, E and F, the extent of FAK phosphorylation was quantified by image analysis and expressed as fold increase with respect to basal FAK phosphorylation observed in Tat-untreated cells. The data are representative of two to four independent experiments that gave similar results. (F) MAE cells adherent to cell culture plates were incubated for 30 minutes at 37°C in the absence or in the presence of 100 ng/ml of GST-Tat. Then, cells were lysed, immunoprecipitated with anti-FAK antibodies, and immunoblotted with anti-FAK or anti-phosphotyrosine antibodies. a, phosphorylated protein; t, total protein.