Anti bcl xl

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)].

Cells were seeded in six-well plates (2.5 × 10⁵/well for 3-h treatment; 2 × 10⁵/well for 24-h treatment), allowed to adhere for 24 h and treated with TRAIL or APG350 for either 3 or 24 h. Whole-cell lysates were prepared using RIPA buffer and analyzed by western blot as described^{23 (link)}. Primary and secondary antibodies used were purchased from: Cell Signaling Technology, Frankfurt/ Main, Germany (anti-caspase-8, anti-PARP, anti-phospho-p38, anti-phospho-IκBα, anti-phospho-p42/44, anti-phospho-JNK, anti-p38, anti-p42/44, anti-JNK, anti-mouse-IgG-HRP, anti-rabbit-IgG-HRP), BD Bioscience, Heidelberg, Germany (anti-Bcl-xL), R&D Systems, Minneapolis, Canada (anti-Bid), Santa Cruz, Heidelberg, Germany (anti-IκBa, anti-goat-IgG-HRP) and Sigma-Aldrich (anti-β-actin).