MafB interacts with c-Fos and inhibits transactivation of NFATc1. (A) BMMs were transduced with pMX-IRES-EGFP (control) or MafB retrovirus and cultured with M-CSF and RANKL for the indicated times. Cells were harvested from each time point and lysates were analyzed for c-Fos and actin by Western blot analysis. (B) 293T cells were cotransfected with NFATc1 6.2-kb promoter luciferase reporter and c-Fos (80 ng) together with the indicated amounts of MafB. Each well was also cotransfected with 20 ng of a β-galactosidase expression vector to control for transfection efficiency. Luciferase activity was normalized to β-galactosidase activity as expressed by the cotransfected plasmid. Data represent the mean and the SE of triplicate samples. Results are representative of at least 3 independent sets of similar experiments. (C) 293T cells were transfected with HA-tagged c-Fos plasmid. After 36 hours of transfection, cell lysates were incubated with glutathione S-transferase (GST) or GST-MafB fusion proteins (2 μg of each) immobilized on glutathione-Sepharose beads. The beads were washed and the bound proteins were resolved by SDS-PAGE and detected by Western blotting (WB) using anti-HA (top panel) and anti-GST (middle panel) antibodies. Whole-cell extracts (WCEs) were also subjected directly to Western blot analysis with the anti-HA antibody to show that equal amounts of c-Fos were expressed (bottom panel). (D) EMSA analysis was performed with ³²P-labeled probes spanning AP1-binding sites in the mouse NFATc1 promoter and c-Fos prepared using TNT rabbit reticulocyte lysate. Specific binding was determined by cold competition using unlabeled wild-type and mutant probes at 1.5-fold and 5-fold molar excess concentrations (lanes 3-6). C-fos lysate and probe were incubated with the indicated amounts of GST or GST-MafB proteins (lanes-7-10). (E) ChIP assay of c-Fos binding to NFATc1 promoter region. BMMs were treated with or without RANKL for 1 day before cross-linking. Samples were immunoprecipitated with control IgG or anti–c-Fos antibody and subjected to PCR amplification with specific primers for AP1-binding sites in the NFATc1 promoter region.