Anti dr5

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).

The human pancreatic cancer cell lines PANC-1 and Suit-2 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). PANC-1 cells were grown in Dulbeccos' Modified Eagle's Media (DMEM; Invitrogen, Carlsbad, CA)) and Suit 2 cells were grown in RPMI 1640 supplemented with penicillin (5 units/mL), streptomycin (5 μg/mL), and 10% heat-inactivated FBS.
DR5 agonist antibody, TRA-8, was generated as previously described [48 ]. All antibodies used were commercially available, including anti-caspase-8 (BD Bioscience, San Jose, CA), anti-caspase-3 (Enzo Life, Plymouth Meeting, PA), anti-FADD and anti-CaM (Millipore, Billerica, MA), Src (Cell Signaling Technology, Danvers, MA), anti-DR5 (Prosci, Poway, CA), anti-c-FLIP (Enzo Life, Farmingdale, NY) and anti-GAPDH (Santa Cruz Biotech, Santa Cruz, CA).
Tamoxifen (TMX) and Trifluoperazine (TFP) were obtained from Sigma-Aldrich (St. Louis, MO). Protein G-agarose and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA). Caspase-8 Inhibitor, Z-IETD-FMK, was from R&D system. The dual-luciferase activity reporter system was purchased from Promega.