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Fig. 3. FAK, RhoA and pp60src 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 pp60src 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.
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vß3-integrin-dependent activation of focal adhesion kinase mediates NF-
B activation and motogenic activity by HIV-1 Tat in endothelial cells
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), GST (
), FN (
) or BSA (
). (B) GM7373 cells were incubated for 1 hour at 4°C with the indicated concentrations of anti-
1-21 (b), GST-TatK49/52/53/55/56/57A (c), GST-Tat-1e (d). (D) HUVE cells or MAE cells were allowed to adhere on plastic coated with 20 µg/ml of GST-Tat (black bars), FN (hatched bars), or BSA (white bars). At the end of incubation, cell adhesion was quantified. In B, data are expressed as the percentage of cells adherent to the different substrata in the absence of antibodies. Each point is the mean ± s.e.m. of three or four determinations in duplicate. (E) GM7373 cell monolayers adherent to the indicate plastic-immobilized proteins or to tissue culture plastic (t.c.p.) were wounded and incubated for 48 hours in culture medium containing 0.4% FCS in the absence (white bars) or in the presence of free GST-Tat (100 ng/ml; black bars), cRGDfV (grey bars) or cRADfV (hatched bars) (both at 3 µM). At the end of incubation the area of the wound was quantified by image analysis. Each point is the mean ± s.e.m. of four to five fields measured in one experiment out of two or three that gave similar results. The results are expressed as percentage of repair in respect to untreated monolayers adherent to immobilized Tat. (F) Representative microphotographs (magnification 50x) of Tat-adherent GM7373 cell monolayers at T0 and 48 hours after the wounding.
B activation by Tat in ECs. (A) GM7373 were allowed to adhere to plastic coated with the indicated proteins or maintained in suspension (s) for 30 minutes. (B,C) GM7373 adherent to cell culture plates were incubated for 1 hour 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). (D) GM7373 cells adherent to cell culture plates were incubated for 1 hour at 37°C with 100 ng/ml of GST-Tat in the absence (-) or in the presence of SN50, SN50M (both at 25 µg/ml), cRGDfV, cRADfV (both at 3 µM), exoenzyme C3 (5 µg/ml), PP2 or PP3 (both at 100 µM). (E) The different clones adherent to cell culture plates were incubated for 1 hour with (black bars) or without (hatched bars) GST-Tat (100 ng/ml). At the end of incubation, cells were lysed and the activation of the indicated NF-