Recombinant tnf

Cells for Western blot analysis were lysed in Lämmli Buffer (Bio-Rad, Hercules, CA, USA) with protease inhibitors (Thermo Fisher, Waltham, MA, USA) and sodium orthovanadate (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Thirty micrograms of total protein were separated on ExcelGels (GE Healthcare) and transferred onto nitrocellulose membranes (Bio-Rad). After blocking, membranes were incubated with primary antibodies [cleaved caspase-3 antibody (0.5 µg/ml, #MAB835; R&D Systems, Minneapolis, MN, USA), phospho-RIPKs 1 (1:100, #65746; Cell Signalling Technology, Cambridge, UK), phospho-RIPK3 (1:200, #ab209384; Abcam, Cambridge, UK), phospho-MLKL (1:500, #91689; Cell Signalling Technology, Cambridge, UK), or glyceraldehyde 3-phosphate dehydrogenase (1:2000, #2118; Cell Signalling Technology, TNF (1 µg/ml, R&D Systems)] overnight at 4 °C. After further incubation with horseradish-conjugated goat-anti-rabbit antibody (1:10,000, #170-6515; Bio-Rad, Hercules, CA, USA), secondary antibodies were visualized with Supersignal West Dura (Thermo Fisher, Waltham, MA, USA) and signals were detected using ChemiDoc System (Bio-Rad). For blocking of the TNF antibody, 1 µg TNF antibody was pre-incubated with 10 µg recombinant TNF (R&D Systems) for 4 h at 4 °C:

Splenocytes isolated from OT-I.CD901.1XIKK2CA^fl/fl xGzB^Cre mice were stimulated with 20 nM OVA peptide for 48 h at a concentration of 1 × 10⁶ cells/ml. TGFβ (R&D Systems, Minneapolis, MN) was then added to a final concentration of 50 ng/ml. At 30 min and 24 h post TGFβ stimulation, cells were harvested for analysis by flow cytometry and western blotting. Splenocytes from CD2rtTA x DN-IKK2 mice were stimulated in vitro at 1 × 10⁶ cells/ml with 10 μg/ml anti-CD3 (clone 145-2C11) and 10 μg/ml anti-CD28 (clone 37.51) (ThermoFisher Scientific, Waltham, MA). Following 24 h of stimulation, cells were divided and incubated in the presence or absence of 125 ng/ml recombinant TNF (R&D Systems, Minneapolis, MN) for 24 additional hours. The cells were again divided and incubated in the presence or absence of 50 ng/ml TGFβ (R&D Systems, Minneapolis, MN). Cells were harvested at 30 min and 24 h post addition of TGFβ and analyzed by flow cytometry.