Fig. 4. Disruption of the interaction with CH-ILKBP inhibits the localization of ILK to focal adhesions. (A-C) Co-immunoprecipitation. The immunoprecipitates were prepared from C2C12 cells expressing GFP-ILK or GFP-tagged ILK mutant in which residues 180-215 were deleted (D215) with anti-GFP antibodies as described in Fig. 3. The GFP-ILK (lane 1) and GFP-D215 (lane 2) 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). The sample loaded on lane 3 was prepared as those of lanes 1 and 2 except that the cell lysates were omitted (to show bands derived from rabbit IgG, which were strong when HRP-conjugated anti-rabbit IgG antibodies were used, such as in panel C). (D,E) Subcellular localization. C2C12 cells expressing GFP-D215 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-D215 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.