Novex wedgewell 4 20 tris glycine mini gels

LECs were lysed in buffer containing 20 mM Hepes (pH 7.4), 150 mM NaCl, 10 mM NaF, 2 mM Na₃VO₄, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1 mM DTT, 1 mM PMSF and protease inhibitor cocktail (Roche). Protein in the cell extract was quantified using protein quantification kit (Bio-Rad, Philadelphia, PA) and 10 μg total protein was run on Novex™ WedgeWell™ 4–20% Tris-Glycine Mini Gels (Invitrogen) and transferred to an Immobilon-P membrane (Bio-Rad). Membranes were probed with anti-p100/p52, NIK, phospho-IKKα/β (Ser176/180) (16A6), IκBα, TRAF2, TRAF3, and GAPDH antibodies. For co-IP assays, 500 μg total protein of cell extract was incubated with 1 μg of anti-mLTβR (5G11b) overnight, followed by 4 h incubation with 25 μL protein G Agarose beads (Sigma-Aldrich). The beads were then washed with lysis buffer and boiled in 2× Laemmli sample buffer (Bio-Rad). Uncropped immunoblotting images are included in Supplementary Fig 9.

Cells were lysed in buffer containing 20 mM Hepes (pH 7.4), 150 mM NaCl, 10 mM NaF, 2 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1 mM DTT, 1 mM PMSF, and protease inhibitor cocktail (Roche). Protein in the cell extract was quantified using a protein quantification kit (Bio-Rad, Philadelphia, PA) and 10 μg total protein was run on Novex™ WedgeWell™ 4–20% Tris-Glycine Mini Gels (Invitrogen) and transferred to an Immobilon-P membrane (Bio-Rad). Membranes were probed with indicated antibodies. Relative band intensities of the blots were measured with ImageJ and normalized to GAPDH.