Anti sharpin antibody

For isolation of the TNFR1-SC, transformed MEFs were stimulated with 3xFlag-2xStrep-TNF at 0.5 μg/mL for 15 min, and controls were left untreated. Cells were subsequently solubilised in lysis buffer (30 mM Tris HCl [pH 7.4], 150 mM NaCl, 2 mM EDTA, 2 mM KCl, 10% Glycerol, 1% Triton X-100, EDTA-free proteinase inhibitor cocktail (Roche, 5056489001) and 1x phosphatase-inhibitor cocktail 2 (Sigma, P5726-1ML) at 4°C for 30 min. The lysates were cleared by centrifugation, and 3xFlag-2xStrep-TNF (0.5 μg/mL/sample) was added to the untreated samples. Subsequently, the lysates were subjected to anti-Flag immunoprecipitation using M2 antibody coupled sepharose beads (Sigma, A2220-5ML) for 16 h. For FADD immunoprecipitation, transformed MEFs were treated with 20 μM zVAD-fmk (Abcam, ab120487) in the presence or absence of 100 ng/mL 6xHis-TNF for 3 h. Cells were lysed as described above and FADD was immunoprecipitated using anti-FADD antibody (Santa Cruz, sc-5559) and protein G Sepharose Beads (GE healthcare, 17-0618-01) at 4°C for 4 h. For Sharpin immunoprecipitation, anti-Sharpin antibody (ProteinTech, 14626-1-AP) was used. For all immunoprecipitations, the beads were washed three times with lysis buffer. Proteins were eluted in 50 μL of LDS buffer (NuPAGE, Invitrogen) containing 50 mM DTT. Samples were analysed by Western blotting.