Cleaved parp asp214

Cells were washed once with cold PBS and upon removal, cells were lysed in radio-immunoprecipitation assay (RIPA) buffer supplemented with PhosSTOP phosphatase and CompleteMini protease inhibitors (Roche, Switzerland) for protein extraction. Proteins were separated by polyacrylamide gel electrophoresis PAGE using 4-12% gradient gels followed by protein transfer onto nitrocellulose membranes with iBLOT2 (ThermoFisher Scientific, Waltham, MA). Membranes were then blocked in 5% nonfat dry milk in Tris- buffered saline with Tween-20 (TBST) and incubated at 4oC with primary antibodies. Proteins of interest were detected with horseradish peroxidase-conjugated secondary antibodies and Advansta WesternBright™ ECL Spray was used for detection (Thomas Scientific, Swedesboro, NJ). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): phospho-RB (Ser780) (#3590), RB (#9309), Cyclin D1 (#2978), phospho-CDK1 (Tyr15) (#4539), CDK1 (#77055), Wee1 (#13084), gamma-H2AX (Ser139) (#80312), H2aX (#7631) and cleaved PARP (Asp214) (#5625). Antibody to p53 (#ab32389) was purchased from Abcam (Cambridge, UK). For loading control, antibody to β-actin (#sc-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Control and treated BxPC-3 cells were harvested by scraping and washing with cold PBS containing protease inhibitors. Cell lysates were prepared in RIPA lysis buffer, as described earlier [21 (link)]. Equal amounts of protein (50 μg) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Primary antibodies against cleaved-PARP (Asp214) (Cell Signaling, Danvers, MA, USA), hENT1 (F-12) and PCNA (F-2) (Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin (Proteintech, Chicago, IL, USA) were reacted at 1:1000 with blots. The HRP-conjugated, anti-rabbit or anti-mouse secondary antibodies (Cell Signaling, Danvers, MA, USA) were used at a dilution of 1:3000. Band expressions were developed using Pierce ECL reagents (Thermo Scientific, Rockford, IL, USA).

Total protein was extracted from gastric cancer cell lines and paired primary tumors and non-tumorous tissues using RIPA lysis buffer with proteinase inhibitor. Protein concentration was measured by the method of Bradford (Bio-Rad, Hercules, CA). Twenty-microgram of protein mixed with 2 × SDS loading buffer were loaded on each lane, separated by 12% SDS-polyacrylamide gel electrophoresis. YY1 protein was then detected using anti-YY1 antibody (1:1000, H-10, sc-7341, Santa Cruz Biotechnology, Dallas, TX). Other antibodies applied included cleaved-PARP (Asp214) (1:1000, #9541, Cell Signaling, Danvers, MA), active-β-catenin (1:1000, #05-665, Millipore, Billerica, MA), β-catenin (1:10000, #610154, BD Transduction Laboratories, San Jose, CA), CCND1 (1:1000, #2926, Cell Signaling, Danvers, MA ), c-Myc (1:1000, #9402, Cell Signaling, Danvers, MA), anti-Mouse IgG-HRP (1:30000, #00049039, Dako, Glostrup, Denmark) and anti-Rabbit IgG-HRP (1:40000, #00028856, Dako, Glostrup, Denmark).

Three TMAs containing ovarian cancer sections were provided by Dr. Tanja Pejovic (Oregon Health and Science University). These TMAs contained section from102 different cases. IHC was performed on paraffin embedded tumors according to the staining procedure provided by the VECTASTAIN ABC KIT using ImmPACT DAB and Hematoxylin QS as a counterstain (Vector Laboratories, Burlingame, CA). Anti-ARID3B IHC Antibody (Bethyl Laboratories, Montgomery, TX), anti-KI67 (Novus Biologicals, Littleton, CO), and Cleaved PARP (Asp214) (Cell Signaling Technology, Danvers, MA) were used. Slides were scanned at the 20x magnification setting using Aperio Scan Scope (Vista, CA). Hematoxylin and eosin (H&E) staining was performed on xenograft tumors from SKOV3IP-RFP and SKOV3IP-RFP cells injections according to standard procedures.

RPMI-1640 medium and fetal bovine serum (FBS) were from Mediatech, Inc. (Manassas, VA, USA), N-acetyl cysteine (NAC), catalase, SOD and HiQ Standard Agarose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glutathione (GSH), DMPO, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and NADPH were obtained from Cayman Chemical (Ann Arbor, MI, USA). FMN was from TCI AMERICA (Portland, OR USA). Antibodies to detect total c-Jun, phosphorylated c-Jun (Ser63), total c-Fos, phosphorylated c-Fos (Ser32), total JNKs, phosphorylated JNKs (Thr183/Tyr185), total p38, phosphorylated p38 (Thr180/Tyr182), total ATR, phosphorylated ATR (Ser428), phosphorylated ATM (Ser1981), total H2AX, phosphorylated H2AX (Ser139), total p53, phosphorylated p53 (Ser15), total CDC2, phosphorylated CDC2 (Tyr15), phosphorylated cyclin B1 (Ser147), caspase 3 (Asp175), PARP, cleaved PARP (Asp214) and iNOS were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibodies to detect total ATM, Bax and p21 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).