Fig. 6. Protein kinase C regulates the formation of the PINCH-ILK-CH-ILKBP complex. (A-C) CH-ILKBP was immunoprecipitated from lysate of human mesangial cells that were treated with calphostin C (1 µM) for 50 minutes (lane 2), or that of untreated human mesangial cells as a control (lane 1) with monoclonal anti-CH-ILKBP antibody 1D4. The immunoprecipitates were analyzed by western blotting with mouse monoclonal anti-CH-ILKBP antibody 3B5 (A), mouse monoclonal anti-ILK antibody 65.1 (B) and rabbit polyclonal anti-PINCH antibodies (C), respectively. The sample loaded on lane 3 was prepared as those of lanes 1 and 2, except that the cell lysates were omitted (to show mouse IgG bands, which were apparent in A and B). Note that treatment of cells with protein kinase C inhibitor calphostin C significantly reduced the amounts of ILK and PINCH associated with CH-ILKBP (compare lanes 1 and 2). Similar results have been obtained with other cell types including human IMR-90 fibroblasts and mouse C2C12 myoblasts (not shown in the figure). (D-F) Human 293 embryonal kidney cells expressing FLAG-ILK was incubated in normal medium (lane 1) or medium containing 0.2 µM calphostin C (lane 2) or 0.4 µM cytochalasin D for 60 minutes. FLAG-ILK was immunoprecipitated from the cell lysates with monoclonal anti-FLAG antibody M2 as described in Materials and Methods. The immunoprecipitates were analyzed by western blotting with mouse monoclonal anti-FLAG antibody M2 (D), mouse monoclonal anti-CH-ILKBP antibody 3B5 (E) and rabbit polyclonal anti-PINCH antibodies (F), respectively. Note that calphostin C (lane 2), but not cytochalasin D (lane 3), inhibited the interactions of FLAG-ILK with CH-ILKBP (E) and PINCH (F). The experiments were performed four times and similar results were obtained.