Anti ciap2

Western blotting was performed on various cancer cell lines to investigate the alteration of protein expression as described previously [35 (link),36 (link)]. The harvested cells were lysed using RIPA lysis buffer and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The protein was transferred onto nitrocellulose membrane (GE Healthcare Life Science, Pittsburgh, PO, USA) and were incubated with primary antibodies overnight, and then the secondary antibody was incubated at room temperature for 2 h. Finally, expression of proteins was detected by an enhanced chemiluminescence kit (Merck Millipore, Darmstadt, Germany). The information on primary antibodies was provided as below: anti-Bcl-2 and anti-DR4 from Abcam (Waltham, MA, USA); anti-Mcl-1 and anti-cIAP2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Bax, anti-Bim, and anti-XIAP from Biosciences (San Jose, CA, USA); anti-survivin from R&D System; anti-Bcl-xL, anti-cIAP1, anti-DR5, anti-PARP, anti-USP2, and anti-cleaved caspase-3 from Cell Signaling Technology (Beverly, MA, USA); anti-c-FLIP and anti-caspase3 from Enzo Life Sciences (San Diego, CA, USA). RT-PCR and quantitative PCR were used to analyze mRNA expression, and primer sequences were described previously [37 (link)].

Whole-cell lysates were obtained from cultured cells with RIPA buffer (Boston Bioproducts Inc., Ashland, MA, USA) containing protease inhibitor cocktail (Roche, Basel, Switzerland) plus 10 mM NaF and 10 mM Na₃VO₄. Proteins were fractionated by 10% SDS-polyacrylamide gel and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were incubated with the following antibodies: Anti-A20 (Active Motif, Carlsbad, CA, USA); Anti-cIAP-1, Anti-cIAP-2, Anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Anti-GAPDH (Millipore); Anti-XIAP (Transduction Laboratories, San Jose, CA, USA); and Anti-vinculin (Sigma). Following incubation of anti-IgG, the proteins were visualized using an ECL detection system (Amersham, Piscataway, NJ, USA).

Human renal carcinoma (Caki-1 and A498), human lung cancer (A549), and human breast cancer (MDA-MB361) were procured from American Type Culture Collection (Manassas, VA, USA). Human recombinant TRAIL, zVAD-fmk, and anti-survivin were provided by the R&D system (Minneapolis, MN, USA). MG132, PD98059, AG-490, compound C, and NVP-BEZ235 were supplied from Calbiochem (San Diego, CA, USA). Dexamethasone, cycloheximide, AR-A014418, PP242, BAY11-7082, rapamycin, and anti-actin were provided from Sigma Chemical Co. (St. Louis, MO, USA). Anti-PARP, anti-Bcl-xL, anti-DR5, anti-cIAP1, anti-caspase-8, anti-phospho-GSK3β, and anti-GSK3β were provided by Cell Signaling Technology (Beverly, MA, USA). Anti-Bim, anti-Bax, and anti-XIAP were obtained from BD Biosciences (San Jose, CA, USA). Anti-Mcl-1, anti-Bcl-2, anti-cIAP2, and anti-Cbl were purchased from Santa Cruz Biotechnology (St. Louis, MO, USA). SB203580, SP600125, and anti-c-FLIP(L) were obtained from Enzo Life Sciences (San Diego, CA, USA). Anti-DR4 were obtained from Abcam (Cambridge, MA, USA). pCMV-Myc-Cbl plasmid was a gift from Dr. S. J. Kim (CHA University, Korea). GSK3betaS9A (1016) was a gift from Scott Friedman (Addgene plasmid # 49492; http://n2t.net/addgene:49492; RRID: Addgene_49492) [36 (link)].