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Fig. 4. DH-dead p115 RhoGEF inhibits formation of stress fibers and focal adhesions and RhoA activity on FN. (A) REF52 fibroblasts were transfected with vector encoding GFP-p115(FL) or GFP-p115(4A). 24 hours post transfection, the cells were lysed, pulldowns performed with RhoA(17A) and samples blotted with an antibody against GFP. (B) REF52 cells transfected with vector encoding GFP-p115(4A) were serum-starved, held in suspension for 2 hours and plated onto FN-coated coverslips. The cells were then fixed and stained with phalloidin to visualize F-actin and an antibody against phosphotyrosine to visualize focal adhesions. Bar, 40 µm. (C) REF52 cells overexpressing p115(4A) were plated onto FN-coated coverslips for 60 minutes. The cells were then treated with either DMSO or 10 µM nocodazole for 30 minutes and fixed and stained with phalloidin to visualize stress fibers. Cells were scored according to whether they had prominent stress fibers versus few to no stress fibers. (D) Cells were transfected with GFP-p115(4A), serum-starved and held in suspension for 2 hours. The cells were then plated onto FN-coated dishes, and GST-RBD pulldowns performed, and samples blotted with an antibody against RhoA to visualize the levels of RhoA activity. Lysates were also blotted with an antibody against GFP to show the levels of expression of GFP-p115(4A).
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