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Fig. 7. GFP-FAK co-immunoprecipitates and localizes with co-expressed Myc-PDZ-RhoGEF and Myc-PDZ-RhoGEF(1-585). (A) HEK293T cells were transfected with with 0.5 µg GFP-FAK and Myc-PDZ-RhoGEF(1-585) or Myc-p115RhoGEF. To measure the amount of FAK in complex with the individual GEF proteins, immune complexes were isolated from lysates using anti-Myc antibody, and subjected to SDS-PAGE and western blotting with anti-FAK (top panel) or anti-GFP (second panel) antibodies. The expression of GEF proteins and GFP-FAK in lysates was determined by SDS-PAGE and immunoblotting lysates directly with anti-Myc or anti-GFP antibodies (bottom two panels). (B) Rat2 cells stably expressing GFP-FAK were transiently transfected with Myc-PDZ-RhoGEF, Myc-PDZ-RhoGEF(1-585) or Myc-p115RhoGEF, detached and seeded on fibronectin coated glass T dishes, fixed and Myc stained visualized using TIRF microscopy. Images show the colocalization of GFP-FAK and Myc-tagged PDZ-RhoGEF (top and middle panels), and the lack of colocalization of GFP-FAK and Myc-tagged p115RhoGEF. (C) Western blot analysis of endogenous PDZ-RhoGEF expression and phase-contrast images of control Rat2 cells and Rat2 cells treated with PDZ-RhoGEF siRNA. (D) Fluorescent images and cell elongation quantification of control Rat2 cells and Rat2 cells treated with PDZ-RhoGEF siRNA.
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