Mouse anti cleaved parp

Cells were harvested, washed with PBS, and resuspended in lysis buffer (50 mmol/L Tris‐HCl [pH 7.6], 150 mmol/L NaCl, 1 mmol/L PMSF, 1 mmol/L DTT, 10 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin A, and 1% NP‐40) with phosphatase inhibitor (10 mmol/L NaF and 1 mmol/L Na₃VO₄) for 20 minutes on ice. After centrifugation, the supernatants were isolated and used as cell lysates. The cell lysates were separated by SDS‐PAGE and transferred onto PVDF membranes. The membranes were blocked with blocking buffer (0.1% casein, 0.1% gelatin, and 0.1% Tween‐20 in TBS) and incubated with the following primary Abs: rabbit anti‐DYRK2 (Sigma‐Aldrich), mouse anti‐p53, c‐Myc, Cyclin D1 and GAPDH and rabbit anti‐Cyclin D2 (Santa Cruz Biotechnology), mouse anti‐phospho‐p53‐Ser46 (Bio Academia), mouse anti‐cleaved PARP, and rabbit anti‐cleaved caspase 3 (Cell Signaling Technology). Membranes were then washed three times in TBS with 0.05% Tween‐20, and incubated with peroxidase‐conjugated anti‐rabbit IgG (Santa Cruz Biotechnology) or peroxidase‐conjugated anti‐mouse IgG κ‐BP (Santa Cruz Biotechnology). Signals were detected using a chemiluminescent regent, ImmunoStar LD (Wako). Signals were observed and band intensity was measured using a Fusion‐Solo system (M and S Instruments).