FIGURE 11.

Role for αvβ3-integrin in Rac1 activity. A, β1-k/d or control cells were pretreated with an αvβ3-integrin inhibitor, LM609 (10 μg/ml), for 1 h and then treated with TGF-β1 or vehicle for 15 min. GTP-bound, active Rac1 was detected by a pulldown assay, and the resulting immunoreactive bands were scanned and analyzed by NIH ImageJ software. Mean ± S.E. of three separate experiments is shown. *, p < 0.05 for effects of the knockdown, †, p < 0.05 for effects of the inhibitors. arb. U, arbitrary units. B, β1-k/d or control HKC cells expressing the Elk-Gal-luc promoter construct, along with β-galactosidase expression vector, were treated with LM609 (10 μg/ml) for 1 h followed by a 24-h incubation with TGF-β1. Resulting luciferase activity, mean ± S.E. of triplicates, after standardization with β1-galactosidase expression is shown. *, p < 0.05 for effects of knockdown, †, p < 0.05 for effects of the inhibitors. C, either β1-k/d or control cells that were treated with another αvβ3-integrin inhibitor, XJ735 (20 μm), for 1 h and lysates were analyzed by immunoblotting with indicated antibodies. Representative blots from at least three separate experiments are shown. p indicates phosphorylation. D, HKC cells transfected with either GFP-integrin or GFP-β3-integrin expression vector were sorted for GFP and replated for Rac assay. Rac activity after treatment with TGF-β1 or vehicle for 15 min was evaluated by Rac G-LISA assay. Mean ± S.E. (n = 3) of relative change from control cells is shown. *, p < 0.05 as compared with GFP-expressing control cells treated with vehicle. T/F, transfection.