Anti bax 2d2

Anti–Bcl-2 (Ab-1) antibody was purchased from Merck Biosciences (Darmstadt, Germany). Anti-Bcl-X_L, anti-Bcl-w, anti-Bid, anti-Bim (C34C5), anti-Bak, anti-caspase-3, anti-caspase-8 (1C12), anti-caspase-9 and HRP-conjugated secondary antibodies were obtained from New England Biolabs (Berlin, Germany). Anti-Mcl-1 (22) antibody was from BD Bioscience (Heidelberg, Germany), anti-Bax (2D2) was from Santa Cruz Biotechnology (Heidelberg, Germany) and anti-cytochrome c (clone 7H8.2C12) was purchased from BD Biosciences (Heidelberg, Germany). Anti-β-actin (AC-15) and anti-α-tubulin (DM1A) antibodies were obtained from Sigma-Aldrich (Munich, Germany). Bax activation was measured via flow cytometry by staining with an antibody specific for the conformation of activated Bax (Clone 3; BD Pharmingen, Heidelberg, Germany). PCR primers and siRNAs were purchased from Eurofins MWG Operon (Ebersberg, Germany).

For Western blot analysis, cells were lysed as previously described [43 (link)-44 (link)]. Protein determination was performed by using the BCA Protein Assay (Thermo Scientific, Rockford, IL). Samples were supplemented with loading buffer (250 mM Tris pH 6.8, 2% SDS, 10% glycerin, 4% beta-mercaptoethanol, 1% bromophenol blue) and boiled for 2 minutes. Equal amounts of protein for each sample were migrated in SDS-polyacrylamide gels and blotted onto nitrocellulose filters, as previously described [43 (link),44 (link)]. The following Abs were used: anti-p53 (DO-1), anti-MDM2 (SMP14), anti-p21 (C-19), anti-PUMAα/β (H-136), anti-PARP-1 (H-250) and anti-Bax (2D2) purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Phospho-p53 (Ser15) and anti-Phospho-p53 (Ser392) from Cell Signaling Technology (Danvers, MA); anti-tubulin from Sigma-Aldrich. After incubation with anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary Abs (Sigma-Aldrich), specific reactions were revealed with the ECL Lightning detection kit (Perkin Elmer, Waltham, MA). Densitometry values for Western blot were estimated by the ImageQuant TL software (GE Healthcare, Buckinghamshire, UK) and were expressed as arbitrary units (a.u.). Multiple film exposures were used to verify the linearity of the samples analyzed and to avoid saturation of the film.