Anti ciap1

Antibodies used for immunoblots were as follows: anti-ASS1 (Polaris, San Diego, CA, USA), anti-LC3 (Sigma), anti-p62 (Sigma), anti-RIP1 (Cell Signaling), anti-caspase 8 (Cell Signaling), anti-BCL-2 (Cell Signaling), anti-cIAP1 (Cell Signaling), anti-cleaved PARP (Cell Signaling), anti-caspase 3 (Imgenex, San Diego, CA, USA), anti-RIP3 (Abcam, Cambridge, MA, USA), anti-Atg5 (Santa Cruz, Dallas, TX, USA), anti-Atg7 (Microbiology Laboratories, Woburn, MA, USA), and anti-actin (Sigma). Cells were lysed in RIPA buffer and protein concentrations were determined by BCA kit (Pierce, Waltham, MA, USA). In all, 25–40 μg of proteins was resolved by NuPAGE (Invitrogen) and transferred onto PVDF membranes (Immobilon-P, Millipore, Darmstadt, Germany). Antibody detection was accomplished using enhanced chemiluminescence (Western Lightning, PerkinElmer, Melville, NY, USA).
For co-IP, SK-LMS-1 cells were treated with PBS, 1 μg/ml ADI-PEG20, 20 μM chloroquine, or both for 3 days. Following treatment, cells were lysed in 0.2% NP-40 buffer and protein concentration was determined by BCA kit (Pierce). Lysates were incubated with anti-RIP1 (Cell Signaling) and protein A/G beads (Pierce). The immunoprecipitates were subsequently immunoblotted with anti-RIP3 (Abcam), anti-RIP1 (Cell Signaling), anti-caspase 8 (Cell Signaling) and anti-actin (Sigma) as described above.

American Type Culture Collection (ATCC) supplied all cancer cells and TCMK-1 cells (Manassas, VA, USA), and Korea Cell Line Bank supplied the human skin fibroblasts (HSF). Cells were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 5% penicillin–streptomycin, and 100 μg/mL gentamycin. Calbiochem supplied N-acetylcysteine (NAC) and trolox (San Diego, CA, USA), and R&D system supplied z-VAD-fmk, recombinant human recombinant TRAIL, and anti-survivin antibodies (Minneapolis, MN, USA). Santa Cruz Biotechnology supplied anti-Mcl-1, anti-cIAP2, anti-Itch, anti-Cbl, and anti-ATF4 antibodies (Dallas, TX, USA), and Cell Signaling Technology provided anti-PARP-1, anti-Bcl-2, anti-Bcl-xL, anti-caspase-3, anti-cIAP1, anti-DR5, and anti-CHOP antibodies (Beverly, MA, USA). Enzo Life Sciences provided anti-c-FLIP and anti-GRP78/94 antibodies (Ann Arbor, MI, USA), and Abcam supplied the anti-DR4 antibody (Cambridge, MA, USA). BD Biosciences supplied the anti-Bim antibody (San Jose, CA, USA). Sigma Chemical Co. supplied other reagents and the anti-actin antibody (St. Louis, MO, USA).

American Type Culture Collection (Manassas, VI, USA) supplied all cell lines, and cells were cultured in Dulbecco’s modified Eagle’s medium consisting of 10% fetal bovine serum, 5% penicillin–streptomycin antibiotic, and 100 μg/mL gentamycin. R&D (Minneapolis, MN, USA) supplied the recombinant human TRAIL, z-VAD, and anti-survivin antibodies. Anti-Mcl-1, anti-STAMBPL1, anti-USP53, and DR5 siRNA was purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and Cell Signaling Technology (Beverly, MA, USA) supplied the anti-PARP-1, anti-Bcl-2, anti-Bcl-xL, anti-caspase-3, anti-cIAP1, anti-c-FLIP, and anti-DR5 antibodies. Anti-DR4 antibody was obtained from Abcam (Cambridge, MA, USA). BD Biosciences (San Jose, CA, USA) supplied the anti-Bim and anti-XIAP antibody. STAMBPL1 plasmid was provided by Dr. Eek-Hoon Jho (University of Seoul, Seoul, Korea). Control (GFP) siRNA was purchased from Bioneer (Daejeon, Korea). Anti-actin antibody and all other reagents were supplied by Sigma Chemical Co. (St. Louis, MO, 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)].

The SCC‐9 and HSC‐3 cell lines were collected after being treated with various doses of hispolon (25, 50, 75, and 100 μM) or vehicle control for 24 h. For Western blot, cells lysates were collected and separated by 10–15% SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). After electrophoresis, the proteins were subsequently transferred onto PVDF membranes (Millipore) and blocked with 5% skimmed milk for 1 h at room temperature. Indicated primary antibodies, including anti‐PARP, anti‐pro‐caspase‐3, ‐8, 9, anti‐cleaved caspase‐3, ‐8, ‐9, anti‐ERK1/2, anti‐phospho‐ERK1/2, anti‐JNK1/2, anti‐phospho‐JNK1/2, anti‐p38 MAPK, anti‐phospho‐p38 MAPK, anti‐HO‐1, anti‐cIAP1 (Cell Signalling Technology), and anti‐β‐actin (Abcam) were then added to probe the membrane overnight. After washed with Tris‐buffer saline‐0.1% Tween‐20 (TBS‐T) and incubated with secondary antibodies (Seracare; 1:10000 dilution), the signals were detected using Luminescent Image Analyser (LAS 4000 mini, GE Healthcare Bio‐Sciences). The relative photographic density was quantified by Image J.

Western blot was performed as described previously [30 (link)]. Antibody concentrations of primary antibodies were used as follows: Anti-DR4 (1:1000; Pro Sci, Fort Collins, CO, USA), anti-caspase-8 (1:1000; Enzo), anti-CDK9, anti-PARP, anti-FADD, anti-Bax, anti-Bak, anti-Mcl-1, anti-Bcl-xL, anti-Bcl-2, anti-cFLIP (AdipoGen, San Diego, CA, USA), anti-cIAP1, anti-cIAP2, anti-XIAP, anti-survivin (1:1000; Cell Signaling Technology), anti-caspase-9 (1:500; Cell Signaling Technology), anti-DR5, anti-Bid (1:2000; Cell Signaling Technology), anti-β-actin (1:5000; Sigma-Aldrich), anti-caspase-3 (1:2000; R&D, Minneapolis, MN, USA), anti-RNA polymerase II total (RNA Pol II) (1:2000), anti-pSer2 RNA Pol II (1:5000; Covance, Princeton, NJ, USA). Immuno-complexes were detected using peroxidase-conjugated anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG (1:5000; Cell Signaling Technology) followed by chemiluminescence detection (Pierce ECL Western Blotting Solution; Thermo Fisher Scientific).