Cisplatin and MK-1775 Effects on Cell Signaling

Cells were treated with cisplatin (1.5 μmol/L), MK-1775 (0.25 μmol/L) either alone or in combination as previously indicated. Cell extracts were prepared and Western blot analysis was conducted as described previously (28 (link)). Membranes were blocked for 1 hour at room temperature using 1% powdered milk in 0.1% Tween-20 in TBS, and incubated overnight with the following primary antibodies, including phospho-γH2AX (Ser139; #2577), phospho-CDC2-Tyr15 (#9111), CDC2 (#9112), cyclin B1 (#4138), phospho-CDC25C-Ser216 (#4901), phospho-Histone H3 (p-HH3, #9701), PARP-1, CHK1 (#2345), and CHK1-Ser345 (#2341), all from Cell Signaling Technology; β-actin (#A5316; Sigma-Aldrich). Membranes were then washed with 0.1% Tween-20 in TBS and incubated for 1 hour at room temperature with species-specific horseradish peroxidase–conjugated secondary antibodies, and protein signals were developed using the SuperSignal West chemiluminescent system (Pierce Biotechnology). Membranes were stripped and reprobed with anti–β-actin to verify equal protein loading.