Anti bcl 2 n 19

Recombinant human TRAIL and anti-His were obtained from R&D Systems (Minneapolis, MN). MG132, propidium iodide (PI), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and anti-Flag-HRP were purchased from Sigma (St. Louis, MO). Rabbit anti-DTX1 polyclonal antiserum against GST-Deltex1 was generated as described^{9 (link)}. The following antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA): anti-caspase-3 (H-277), anti-DR4 (H130), anti-DR5 (N-19), anti-Mcl-1 (S-19), and anti-Bcl-2 (N-19). Annexin V-Cy5 was obtained from Biovision (Mountain View, CA). Anti-Myc (9B11), anti-caspase-8 (1C12), and anti-active caspase-3 (D175) were purchased from Cell Signaling (Beverly, MA). Anti-actin (clone C4) and anti-β-tubulin (clone AA2) were purchased from Millipore (Temecula, CA). Anti-human FLIP mAb (NF6) was purchased from AdipoGen (San Diego, CA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. WesternBright ECL HRP substrate was obtained from Advansta Corporation (Menlo Park, CA). Dapi-Fluoromount-G^TM was obtained from SouthernBiotech (Birmingham, AL). Protein G Mag Sepharose^TM Xtra was obtained from GE Healthcare (Piscataway, NJ).

Cells were lysed with T-PER® Tissue Protein Extraction Reagent (# 78510; Pierce Biotechnology, Rockford, IL) added with protease inhibitors. Lysates were separated on pre-cast gels (Bio-Rad Laboratories; Hercules, CA) and immunoblotted using standard procedures. Anti-p21 (C19) (# sc-397; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bax (N20) (# sc493, Santa Cruz Biotechnology), anti Bcl-2 (N19) (# sc-492, Santa Cruz Biotechnology), anti-Gli1 (# NB600-600, Novus Biologicals, Littleton, CO, USA) and HRP-conjugated secondary antisera (Santa Cruz Biotechnology) were used followed by enhanced chemiluminescence (ECL Amersham, Amersham, UK). Densitometry calculations for western blot were calculated using ImageJ software, verifying for non-saturation and subtracting background. Band intensities were sampled three times and normalized against HSP90 (# 4874; Cell Signaling Technology Inc., Danvers, MA).