Antibodies: ABIN-1 (SC134660, Santa Cruz Biotechnology, Santa Cruz CA or 4664, Cell Signaling, Beverly MA), IκBα (SC371, Santa Cruz Biotechnology), α-tubulin (ab40742, Abcam, Cambridge MA) and β-actin (ab49900, Abcam). Secondary Abs were from Thermofisher. Inhibitors: MG132 (474790–100UG, 20μM), staurosporine (569396, 100nM), and IKK inhibitor VII (401486–1MG, 10µM EMD Millipore-Sigma, Billerica MA).
HEK293T cells were cultured in DMEM and ST2 cells and MEFs were cultured in α-MEM (Sigma, St Louis MO) containing 10% fetal bovine serum (FBS) with L-glutamine and antibiotics (Invitrogen). Transfections were performed with Fugene HD (Promega, Madison WI) or Lipojet (SignaGen, Rockville MD). Plasmids expressing ABIN-1, the ABIN-1 promoter and the Lcn2 promoter were previously described (13 (link)–15 (link)). Luciferase assays were performed as described (7 , 14 (link)). SiRNAs were from Dharmacon (GE, Lafayette CO): Tnip1 (ABIN-1) (L-047652–01), TNFαip3 (A20) (L-058907–02) and non-targeting mock (D-001810–10-20). siRNA transfections were performed 48 h prior to stimulation. In all cases efficiency of knockdown was confirmed by qPCR.
HEK293T cells were cultured in DMEM and ST2 cells and MEFs were cultured in α-MEM (Sigma, St Louis MO) containing 10% fetal bovine serum (FBS) with L-glutamine and antibiotics (Invitrogen). Transfections were performed with Fugene HD (Promega, Madison WI) or Lipojet (SignaGen, Rockville MD). Plasmids expressing ABIN-1, the ABIN-1 promoter and the Lcn2 promoter were previously described (13 (link)–15 (link)). Luciferase assays were performed as described (7 , 14 (link)). SiRNAs were from Dharmacon (GE, Lafayette CO): Tnip1 (ABIN-1) (L-047652–01), TNFαip3 (A20) (L-058907–02) and non-targeting mock (D-001810–10-20). siRNA transfections were performed 48 h prior to stimulation. In all cases efficiency of knockdown was confirmed by qPCR.
