Fig. 7. The F438A point mutation does not inhibit the interactions with PINCH and CH-ILKBP but impairs the localization of ILK to focal adhesions. (A-C) ILK complex formation. Lysates of C2C12 cells expressing GFP, GFP-ILK or GFP-ILK F438A point mutant were mixed with rabbit anti-GPF antibodies. The GFP (lane 4), GFP-ILK (lane 5), GFP-F438A (lane 6) immunoprecipitates were analyzed by western blotting with HRP-conjugated anti-GFP antibodies (A), mouse monoclonal anti-CH-ILKBP antibody 3B5 and HRP-conjugated anti-mouse IgG antibodies (B), or rabbit anti-PINCH antibodies and HRP-conjugated anti-rabbit IgG antibodies (C). Lanes 1-3 were loaded with cell lysates (10 µg/lane) as indicated in the figure. (D,E) Subcellular localization. C2C12 cells expressing GFP-F438A were plated on fibronectin coated coverslips and stained with a mouse monoclonal anti-paxillin antibody and a Rhodamine Red^TX-conjugated anti-mouse IgG antibody. GFP-F438A and paxillin were visualized under a fluorescence microscope equipped with GFP (D) and rhodamine (E) filters. The data shown are representative of three experiments. Bar, 10 µm.