Jun d

Cells were treated with 250 nM ACBI1, cis-ACBI1, or matched DMSO control for 24 h and then harvested for protein lysate preparation. To make lysates, cold lysis buffer containing 5 mM EDTA, 150 mM NaCl, 150 mM Tris, pH 8.0, 1% Triton X-100, 0.001M PMSF, and 1X Roche protease inhibitor cocktail was used to collect the cells after treatment. The cells were sonicated for 15 s at 25% power and centrifuged to remove cellular debris. To determine protein concentration, the BioRad Bradford assay was used with bovine serum albumin as a standard protein. Then, 15–30 μg of protein per sample was separated by SDS-PAGE and then transferred to a PVDF membrane (PerkinElmer, Shelton, CT, USA). Membranes were placed in 5% milk made in TBS-T (150 mM NaCl, 50 mM Tris, 0.1% Tween-20) to block unspecific interactions. The antibodies used for immunoblotting were BRG1 (Cell Signaling, 49360, Danvers, MA, USA), GAPDH-HRP (Cell Signaling, 8884), JUN (Cell Signaling, 9165), JUNB (Cell Signaling, 3753), and JUND (Cell Signaling, 5000). The Clarity ECL substrate (BioRad, Hercules, CA, USA) was used to visualize bands on a Bio-Rad Chemidoc MP instrument.

LN cells of three mice per group and nuclear extracts probed by electro-mobility shift assays to analyse DNA binding of AP-1 or NFκB, as previously described (D'Acquisto et al., 2007 (link)). Briefly, nuclear extracts (3–5 μg) were incubated with 2 μg of poly (dI:dC) in 20 μl of binding buffer with ³²P end-labelled, double-stranded oligonucleotide probes (5×10⁵ cpm), and fractionated on a 6% polyacrylamide gel (29:1 cross-linking ratio) in 0.5% TBE for 2.5 h at 150 volts. Double–stranded oligonucleotide probes were from Promega, UK. Supershift EMSA were performed as above, additionally using the following antibodies: anti-cFos, -FosB, -JunB, -JunD (all Cell Signaling Technology)