Parp sc 8007

Primary antibodies against phospho-EGFR^tyr1068 (CST-3777), total-EGFR (CST-2232), phospho-ERBB2^tyr1248 (CST-2247), phospho-ERBB3^tyr1222 (CST-4784), total-ERK1/2 (CST-9102), phospho-AKT^ser473 (CST-9271), total-AKT (CST-9272), phospho-S6^ser240/244 (CST-5364), total-S6 (CST-2217), phospho-ER^ser167 (CST-5587), phospho-Rb^ser807/811 (CST-8516), CDK4 (CST-2901), and cyclinD1 (CST-2922) were purchased from Cell Signaling, Inc.; total-ERBB3 (sc-415), ER-alpha (sc-8002, F-10), and PARP (sc-8007) were purchased from Santa Cruz; phospho-ERK1/2 and α-tubulin (T-9026) were obtained from Sigma; and total-ERBB2 was obtained from Millipore. Secondary antibodies (anti-mouse and anti-rabbit horseradish peroxidase) were obtained from Dako. 17β-Oestradiol (E2) and 4-hydroxytamoxifen (4-OHT) were purchased from Sigma, and fulvestrant (referred to as ICI) was obtained from Trocis, UK. Neratinib (PB272) was provided by Puma Biotechnology and Pfizer. Everolimus (RAD001) was purchased from Selleck. All chemicals, unless otherwise stated, were purchased from Sigma, UK. All tissue culture grade plastics were obtained from Nunc, UK.

For Western blotting, cells were extracted using RIPA buffer (0.01 M sodium phosphate (pH 7.2), 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (Roche, Indianapolis, IN). Equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membrane was blocked with 1% BSA, and incubated with anti-EpCAM (1 μg/ml), biotinylated polyclonal goat anti-human EPCAM (R&D Systems BAF960), biotin-conjugated anti-human CD326 (clone 9C4, Biolegend #324316), anti-human epithelial cell antigen-specific antibody (clone VU-1D9; Sigma), anti-EpICD mAb (1:100 dilution; 1144-1; Epitomics, Burlingame, CA), PARP (sc-8007, Santa Cruz Biotechnology, INC), or Caspase3 (Asp175, 5A1E, Cell Signaling) mAbs overnight. Membranes were then incubated with HRP-conjugated secondary antibodies (1:5000, Jackson Immunoresearch) for 1 hour at room temperature, and protein expression was detected using an ECL kit (Millipore, Temecula, CA, USA).