Fig. 3. The PH-like motif is not required for the ILK complex formation and localization to focal adhesions. (A-C) ILK complex formation. Lysates of C2C12 cells expressing GFP-ILK, GFP-tagged ILK mutant in which residues 180-190 within the PH-like motif were deleted (dPH) or GFP alone were mixed with rabbit anti-GPF antibodies. The GFP-ILK (lane 4), GFP-dPH (lane 5) and GFP (lane 6) 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 1-3 were loaded with cell lysates (15 µg/lane) as indicated in the figure. (D,E) Subcellular localization. C2C12 cells expressing GFP-dPH 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-dPH and paxillin were visualized under a fluorescence microscope equipped with GFP (D) and rhodamine (E) filters. The experiments were performed twice and similar results were obtained. Bar, 10 µm.