Fig. 1. The Q40A point mutation within the LIM1 domain abolishes the ILK binding and the localization of PINCH to cell-ECM adhesion sites. (A) Three-dimensional structure of the PINCH LIM1 domain. Amino acid residues that comprise the PINCH binding site are labeled. (B,C) Complex formation with ILK. Lysates of C2C12 cells expressing GFP-tagged wild-type or mutant (Q40A) form of PINCH were mixed with rabbit anti-GPF antibodies. The GFP-PINCH (lane 3) and GFP-Q40A (lane 4) immunoprecipitates were analyzed by western blotting with HRP-conjugated anti-GFP antibodies (B) or mouse monoclonal anti-ILK antibody 65.1 and HRP-conjugated anti-mouse IgG antibodies (C). Lanes 1-2 were loaded with cell lysates (13 µg/lane) as indicated in the figure. (D-G) Subcellular localization. C2C12 cells transfected with expression vectors encoding GFP-Q40A (D,E) or GFP-PINCH (F,G) were plated on fibronectin-coated coverslips and stained with a mouse monoclonal anti-paxillin antibody (as a marker of focal adhesions) and a Rhodamine Red^TX-conjugated anti-mouse IgG antibody. GFP-Q40A, GFP-PINCH and paxillin were visualized under a fluorescence microscope equipped with GFP (D,F) and rhodamine (E,G) filters. The experiments were performed three times and similar results were obtained. Bar, 10 µm.