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Fig. 6. Ectopic expression of PDZ-RhoGEF and RhoA-effector-domain mutant rescues elongation induced by knockdown of FAK expression. (A) NIH3T3 cells were transiently transfected with Myc-PDZ-RhoGEF, Myc-p115RhoGEF and Myc-PDZ-RhoGEF(1-585). Twenty-four hours after transfection, lysates were prepared and blotted with anti-Myc antibody. (B) Quantification of cell elongation. The shape of FAK-siRNA-treated cells that express different RhoGEF constructs was determined by measuring the elongation factor for individual cells. (C) FAK-shRNA-treated cells were transiently transfected with control non-functional PDZ-RhoGEF(561-585)-GFP, G14VRhoA/F39A-GFP or G14VRhoA/F39V-GFP. Twenty-four hours after transfection, lysates were prepared and blotted using anti-GFP antibody. The expression of GFP-tagged proteins was determined by immunoblotting individual lysates with anti-GFP and with anti-ERK antibodies to ensure equal loading of lysate samples. (D) Quantification of cell elongation in FAK-shRNA-treated cells expressing different RhoA-effector-domain mutants.
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