Anti dr4

Cells were lysed in lysis buffer (50 mM Tris-HCl, 1% SDS, 2 μg/mL leupeptin, 2 μg/mL aprotinin, 0.5 mM PMSF, and 1 mM DTT). The lysate was sonicated and centrifuged at 15,000 g for 20 min at 4°C, and the supernatant was collected. The protein extract was loaded onto a 7.5, 10, or 12.5% SDS-polyacrylamide gel for electrophoresis, and blotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Rabbit polyclonal anti-DR4, anti-DR5 (Prosci, Poway, CA, USA), and anti-CDK4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit monoclonal anti-Bim (Epitomics, San Diego, CA, USA), and mouse monoclonal anti- cyclin D1 (MBL, Nagoya, Japan) and anti-β-actin (Sigma) antibodies were used as the primary antibodies. The blots were incubated with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ, USA), and signals were detected using a Chemilumi-one chemiluminescent kit (Nacalai Tesque, Kyoto, Japan).

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).

KillerTRAIL (human recombinant) was purchased from Alexis Biochemicals. The nine most highly selective molecules targeting cFLIP were obtained from NCI–DSCB (National Cancer Institute-Drug Synthesis and Chemistry Branch, Rockville, MD, USA). For Western blotting (WB) experiments, we used the following antibodies: anti-FLIP antibodies DAVE2 and NF6 (Adipogen, San Diego, CA, USA), caspase-3 (8G10; OZYME), PARP (Asp214, 19F4; OZYME, Saint-Cyr-l’École, France), caspase-8 (1C12; OZYME), anti-MBP (New England Biolabs, Ipswich, MA, USA) and anti-His (C-ter) (Invitrogen, Carlsbad, CA, USA). Anti-His (ab81663) was used to immunoprecipitate the DISC complex, and the following antibodies were used for WB analysis: anti-Flip (DAVE II, Adipogen), anti-FADD (556402, BD, San Jose CA, USA), anti-casp8 (5F7, EnzoLife, Farmingdale, NY, USA), anti-DR4 (1139, ProSci, Poway, CA, USA) and anti-DR5 (3696, Cell Signaling, Danvers, MA, USA). Anti-rabbit and anti-mouse HRP-linked secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and β-actin (Sigma Aldrich, St. Louis, MO, USA) were also used.