Immortalized TNFR1/2−/− MEFs were obtained from D. Männel (Regensburg) and maintained in RPMI 1640 medium (Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% GlutaMAX (Thermo Fisher Scientific), and 1% penicillin-streptomycin (Life Technologies Inc.) at 37°C with 5% CO2. Human cervical carcinoma cells (HeLa) and human embryonic kidney 293T Phoenix-ECO Ecotropic packaging cells were obtained from and authenticated by the American Type Culture Collection. HeLa and Phoenix-ECO 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% sodium pyruvate (Life Technologies Inc.) at 37°C with 5% CO2. All cell lines were regularly monitored for Mycoplasma infection. Recombinant human TNFα was purchased from PeproTech and Biochrom. BV6 was a gift from D. Vucic (Genentech Inc.), and zVAD.fmk was purchased from Bachem. All other chemicals were obtained from Sigma-Aldrich or Carl Roth, unless indicated otherwise. Antibodies used were monoclonal mouse anti-human vinculin (V9131, Sigma-Aldrich), anti-human IĸBα (#9242, Cell Signaling Technology), anti–phospho-IĸBα (Ser32, 14D4, 2859, Cell Signaling Technology), anti–NF-κB p65 (C-20, sc-372, Santa Cruz Biotechnology), mouse anti-TNFR1 H-5 (sc-8436, Santa Cruz Biotechnology), and rabbit anti-TNFR1 C25C1 (#3736S, Cell Signaling Technology). We note that anti-TNFR1 C25C1 also targets a truncated isoform of TNFR1 in some cell lines, as reported by the company.

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