Anti dr4 antibody

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).

Cells were seeded in 6-well plates at a density of 500,000 cells/well overnight. The next day, 10 μM ATS/DHA was added to HCT116 and DLD-1. After 24 h, cells were harvested and washed with FACS buffer (PBS with 2% FBS).
Presence of DR4 and DR5 receptors on the cell surface of HCT116 and DLD-1 human colorectal cancer cells was determined by FACS. Cells were collected and resuspended in FACS buffer. Then, they were washed and incubated with anti-DR4 antibody (Abcam, Cambridge, UK) or anti-DR5 antibody (Exbio, Praha, Czech Republic). After washing again, the fluorescein (FITC) conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cambridge, UK) was incubated with cells on ice for 1 h. Isotype control staining was performed with mouse IgG (Dako, Glostrup, Denmark). Death receptor expression was measured using the FACS Calibur flow cytometer (BD Biosciences), and data were analyzed by FlowJo v10 (FlowJo, LCC, Oregon, OR, USA).