Anti cleaved parp d214

Total cell lysates were prepared in 2× sample loading buffer (250 mM Tris-HCl (pH 6.8), 10% glycerol, 4% sodium dodecyl sulfate (SDS), 2% β-mercaptoethanol, 0.006% bromophenol blue, 5 mM sodium orthovanadate, and 50 mM sodium fluoride; Bio-Rad, Hercules, CA, USA). Protein concentrations were quantified through the BCA method [24 (link)] using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (5–20 µg) were separated using 6–13% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA (Sigma-Aldrich) and probed with anti-phospho-STAT3 (Y705), anti-STAT3, anti-cyclin E, anti-CDK2, anti-cyclin D1, anti-CDK4, anti-CDK6, anti-survivin, anti-cleaved caspase-9, anti-cleaved caspase-3, anti-cleaved PARP (D214), and anti-vimentin antibodies (Cell Signaling Technology, Beverly, MA, USA); anti-β-actin horseradish peroxidase antibody (Santa Cruz Biotechnology, Dallas, TX, USA); anti-E-cadherin and anti-N-cadherin antibodies (BD Biosciences, San Jose, CA, USA); or anti-Ki-67 antibody (Abcam, Cambridge, UK). The blots were detected using the WEST-Queen detection system (iNtRON Biotechnology, Seongnam, Korea) [25 (link)].

Culture media, fetal bovine serum, antibiotic-antimycotic solution (penicillin 10,000 units/mL, streptomycin 10,000 µg/mL, amphotericin B 25 µg/mL), and trypsin-EDTA solution (1×) were purchased from Gibco (Grand Island, NY, USA). Gefitinib (CAS No. HY-50895) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Trichloroacetic acid (TCA), sulforhodamine B (SRB), crystal violet solution, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-aurora kinase A (#14475), anti-p-aurora kinase A (T288) (#3079), anti-p21 Waf1/Cip1 (#2947), anti-cdc2 (#9116), anti-cdc25C (#4688), anti-PARP (#9532), anti-Cleaved PARP (D214) (#5625), anti-Caspase-8 (#9746), and anti-Cleaved Caspase-8 (D374) (#9496) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).