Rabbit anti p p53 ser 15

Caffeine, an ATM kinase specific inhibitor, okadaic acid for inhibiting PP2A, 3,3’-iminodipropionitrile (IDPN) for vimentin disruption, and propidium iodide (PI) for cell staining were purchased from Sigma-Aldrich (St. Louis, USA). Proteasome inhibitor MG132 was from Calbiochem (San Diego, USA). Etoposide was used to stimulate DNA damage, while nocodazole was used to arrest cells in M phase. These chemicals were from EMD Biosciences, Inc. (San Diego, USA). The p17 monoclonal antibodies were produced previously by our laboratory. Rabbit anti-p-ATM (Ser 1981), rabbit anti-ATM, mouse anti-Chk1, mouse anti-p-Chk1, mouse anti-Chk2, mouse anti-p-Chk2, rabbit anti-CDC25C, mouse anti-CDK1, rabbit anti-p-vimentin (Ser 56), rabbit anti-p-vimentin (Ser 82), rabbit anti-vimentin, rabbit anti-p-p53 (Ser 15), rabbit anti-p53, rabbit anti-p-Plk1 (T210), rabbit anti-Plk1, rabbit anti-Myt1 were from Cell Signaling Technology (Danvers, USA). Rabbit anti-beta-actin antibody was from Millipore (Billerica, MA). Rabbit anti-p-CDC25C (Ser 216), rabbit anti-p-CDK1 (T14 and Y15), rabbit anti-nucleoporin Tpr, rabbit anti-p-p21 (T145), rabbit anti-p21 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-p-Myt1 (T495) antibody was from Sigma-Aldrich. Anti-mouse IgG (H+L) and anti-rabbit IgG (H+L) antibodies were purchased from Kirkegard & Perry Laboratories (Washington, DC., USA).

Whole cell lysates were prepared using a modified RIPA buffer (20 mmol/L Tris-HCl (pH 7.5) 350 mmol/L NaCl, 1 mmol/L Na2EDTA, 1 mmol/L EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 1 mg/mL leupeptin). Samples were supplemented with complete protease inhibitor (Roche) and were sonicated followed by centrifugation. Supernatants were recovered and used for Western blotting according to standard procedures. Primary antibodies: mouse anti-actin (clone AC-74, Sigma, A2228), mouse anti-γH2AX (ser139) (JBW301, Millipore, 06-536), rabbit anti-p-p53 (ser15) (Cell Signalling, 9284), rabbit anti-Cleaved caspase-3 (Asp 175) (Cell Signalling, 9661), mouse anti-PARP-1 (Calbiochem, AM30), rabbit anti-PARP (Cell Signaling, 9542), rabbit anti-p21 (Cell Signaling, 2947), rabbit anti-p21 (C-19, Santa Cruz, 397), mouse anti-α-tubulin (Calbiochem, DM1A), rabbit anti-Caspase-3 (Cell Signaling, 9662). After extensive washing primary antibodies were incubated with the specific secondary antibodies bound to the HRP enzyme (horseradish peroxidase-conjugated antibody) (GE Healthcare) and subjected to chemiluminescence using the ECL system (GE Healthcare).