Fig. 5. The formation of the PINCH-ILK-CH-ILKBP complex precedes cell adhesion and spreading. CH-ILKBP was immunoprecipitated from C2C12 cells that were maintained in suspension (lane 1) or had adhered and spread on fibronectin for 0.5 hour (lane 2), 1 hour (lane 3) or 20 hours (lane 4) 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 anti-PINCH antibodies (C), respectively. Lane 5 was loaded with a sample that was prepared as those of lanes 1 and 2 except that the cell lysates were omitted. Note that PINCH and ILK formed a complex with CH-ILKBP before cells were in contact with fibronectin (lane 1). The experiments were performed three times and similar results were obtained.