Xiap 610763

After 48 h from Myc-tagged XIAP or Myc-tagged eGFP plasmids transfection HEK293-FT cells were pre-treated with FC2 at a final concentration of 20 µM. A parallel set of cells was left untreated as control. After 30 min, cells were treated with TNF-α (50 ng/ml). After further 30 min, cells were harvested, washed with ice-cold PBS and lysed in E1A lysis buffer (ELB; 150 mM NaCl, 50 mM Hepes, pH 7.5, 5 mM EDTA, 0.5% NP40) supplemented with the mix of protease inhibitors at 4 °C for 30 min. Samples were clarified at 4 °C by centrifugation for 10 min at 13,000 rpm and then quantified with Bradford Assay. 1 mg of total protein extracts were incubated overnight at 4 °C with Protein-A-Sepharose (Sigma-Aldrich) and anti-Myc-Tag antibody #2278 (dilution 1:200, Cell Signaling Technology, Danvers, MA, USA). The Protein-A-Sepharose was previously washed with ELB buffer three times. The bound proteins and 50 µg of each lysate were analyzed by SDS-PAGE and immunoblotting with anti-human primary antibodies recognizing: XIAP #610763 (BD Biosciences, Franklin Lakes, New Jersey, USA), TAB1 #3226, TAK1 #5206 (Cell Signaling Technology, Danvers, MA, USA) and Vinculin #V9131 (Sigma-Aldrich).

The PARP inhibitor, olaparib and PLK1 inhibitor, volasertib were obtaind from Selleck Chemicals (Houston, TX, USA). Antibodies against PLK1 (4513), Bcl-xl (2762), Bcl-2 (2876), cleaved caspase-3 (9664), PARP (9542), CD133 (64326), CD44 (3570), NANOG (4903) and GAPDH (5174) were procured from Cell Signaling Technology (Danvers, MA). Antibodies against Caspase-9 (sc-17784) and caspase-3 (sc-56053) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Caspase-8 (51-8084) and XIAP (610763) antibodies were procured from BD Pharmingen (San Diego, CA, USA). Annexin V was obtained from Thermo Fischer Scientific (Waltham, MA).