MafB associates with Mitf and NFATc1 and inhibits their transactivation. (A,D) 293T cells were cotransfected with OSCAR 1.7-kb promoter luciferase reporter and 80 ng of Mitf (A) or 100 ng of NFATc1 (D) together with the indicated amounts of MafB. The data were normalized and presented as described in Figure 4. Results are representative of at least 3 independent sets of similar experiments. (B,E) 293T cells were transfected with Flag-tagged Mitf (B) or HA-tagged NFATc1 (E) plasmid. Western blot analysis using anti-Flag (B) or anti-HA (E) antibodies was performed as described in Figure 4. (C,F) EMSA analysis was performed with ³²P-labeled probes spanning E-box (C) or NFATc1-binding sites (F) in the mouse promoter regions for OSCAR and Mitf (C) or NFATc1 (F) prepared using TNT rabbit reticulocyte lysate as described in Figure 4. Specific binding was determined by cold competition using unlabeled wild-type and mutant probes at 5-fold and 50-fold molar excess concentrations (C,F lanes 3-6). (G) 293T cells were cotransfected with an OSCAR reporter plasmid together with the indicated plasmids expressing Mitf, PU.1, NFATc1, or MafB (150-300 ng). The data were normalized and presented as described in Figure 4. Results are representative of at least 3 independent sets of similar experiments. (H) ChIP assay of NFATc1 binding to the OSCAR promoter region. BMMs were treated with or without RANKL for 2 days before cross-linking. Samples were immunoprecipitated with control IgG or anti-NFATc1 antibodies and subjected to PCR amplification with primers specific for the NFATc1 binding sites of the OSCAR promoter region. Data represent mean ± SD of triplicate experiments.