Fig. 2. The D31A point mutation disrupts the interaction with PINCH and impairs ILK localization to focal adhesions. (A-C) PINCH binding. Lysates of C2C12 cells expressing GFP-tagged wild-type or mutant (D31A) form of ILK were mixed with rabbit anti-GPF antibodies. The GFP-D31A (lane 3) and GFP-ILK (lane 4) immunoprecipitates were analyzed by western blotting with HRP-conjugated anti-GFP antibodies (A), rabbit anti-PINCH antibodies and HRP-conjugated anti-rabbit IgG antibodies (B), or mouse monoclonal anti-CH-ILKBP antibody 3B5 and HRP-conjugated anti-mouse IgG antibodies (C). Lanes 1 and 2 were loaded with cell lysates (10 µg/lane) as indicated in the figure. (D,G) Subcellular localization. C2C12 cells transfected with expression vectors encoding GFP-D31A (D,E) or GFP-ILK (F,G) 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-D31A, GFP-ILK and paxillin were visualized under a fluorescence microscope equipped with GFP (D,F) and rhodamine (E,G) filters. The experiments were performed twice and similar results were obtained. Bar, 10 µm.

Return to article