Figure 6.

Effect of cisplatin on inhibiting 4E-BP1 phosphorylation. (A) Confluent C2C12 cells were treated with IGF-II (25 ng/ml), cisplatin (25 µM), IGF-II together with cisplatin, and rapamycin (100 nM) for 24 hours. A total of 300 µg of cell lysates were immunoprecipitated with phospho-4E-BP1 (Ser65) antibody followed by Western blot analysis as described in Materials and Methods. (B) Confluent C2C12-1.1 and C2C12-2.7 cells were treated with cisplatin (25 µM) for 24 hours. Protein extracts (100 µg/lane) were separated on 10% SDS-PAGE, and subjected to Western blot analysis. Blotting with antibody against actin was used to demonstrate equal protein loading in each lane. Similar results were achieved in two separate experiments.