FIGURE 10.

Rac1 activity in β1-k/d cells and its role in the enhanced ERK and collagen promoter activity. A, cell lysates of β1-k/d or control HKC cells treated with TGF-β1 or vehicle for 15 min were subjected to a pulldown assay, and the GTP-bound form of Rac1 was detected by immunoblotting with antibody to Rac1 (upper panel). A 5-min treatment with EGF shows that cells are capable of activating Rac1 given a known stimulus. Lysates incubated with GDP or non-hydrolyzable GTPγS are shown as negative or positive control, respectively. Equal input of Rac1 protein was verified with whole cell lysates (bottom panel). B, Rac1 activation in TRIPZ-β1-k/d cells was evaluated with increasing duration of doxycycline (DOX) treatment that induces shRNA to β1-integrin in HKC cells. C, Rac1 cellular localization was evaluated with β1-k/d or control HKC cells plated on gelatin-coated glass coverslips and stained with an antibody to Rac1. Membrane localization of the protein is depicted with arrows. Bar = 10 μm. 63× oil objective. D, a Rac1 or negative control shRNA expression vector was transiently transfected in either β1-k/d or control cells along with either Elk-Gal-luc reporter system (left) or -0.4COL1A2-luc construct (right). Reporter activities after a 24-h treatment with TGF-β1 or vehicle were assayed in triplicate, and data from one of three independent experiments are shown as mean ± S.E. of luciferase readings corrected for β1-galactosidase expression. *, p < 0.01 for effects of the knockdown as compared with control cells, †, p < 0.05 for effects of inhibitors as compared with β1-k/d without the shRNA. arb. U, arbitrary units.