Tnf r1 fc

HCT-116 were from ATCC. HCT116 (Bax/Bak)^−/− cells were a gift from R. Youle (NIH, Bethesda, MD), MEF and MEF (Bax/Bak)^−/− cells were a gift from S. Oakes (University of California, San Francisco, CA). Cells were authenticated and regularly mycoplam tested. TRAIL was provided by C.R. Rodrigues dos Reis (University of Groningen, Netherlands). Tunicamycin was purchased from Alexis (San Diego, CA), bortezomib from Millenium Pharmaceuticals (Cambridge, MA), MG-132 from Biomol (Plymouth Meeting, PA, USA), MnTBAP, 3-MA, 5-FU, Suc-LLVY, cisplatin, and necrostatin-1 from Calbiochem, z-VAD-fmk from Bachem (St. Helens, UK), Hoechst 33342 from Immunochemistry (UK), Aggresome Staining Kit, and TRAIL-R1:Fc (ALX-522-004-C050), TRAIL-R2:Fc (ALX-522-005-C050), and TNF-R1:Fc (ALX-522-013-C050) neutralizing antibodies from Enzo Life Sciences (Exeter, UK), Coomassie PLUS (Bradford) protein assay kit and Pierce BCA protein assay kit from Thermo Fisher Scientific, RPMI1640 medium, Q-VD-OPh, rotenone, epoxomicin, propidium iodide, ferrostatin-1, necrosulfonamide, FBS and all other chemicals were purchased from Sigma-Aldrich. Q-VD-OPh was added in concentrations of up to 20–50 µM to fully prevent caspase processing and substrate cleavage, where required [6 (link), 7 (link)].

All chemicals and reagents used were obtained from Sigma-Aldrich (St. Louis, MO, USA) except as indicated. Recombinant receptors (CD40:Fc, OX40:Fc, BAFFR:Fc, and TNF-R1:Fc) and FLAG-tagged ligands (CD154, OX40L, BAFF, and TNF-α) were obtained from Enzo Life Sciences (San Diego, CA, USA). CD40L used in the thermal shift assay was from Peprotech (Rocky Hill, NJ, USA). The monoclonal anti-human CD154 (clone 40804) was obtained from R&D Systems (Minneapolis, MN, USA).