Ids down-regulate the gene expression of OSCAR through interaction with Mitf, as well as that of NFATc1. (A) ³⁵S-labeled mock, Id1, Id2, or Id3 protein was incubated with Flag-Mitf immobilized on M2-sepharose beads, respectively. After washing, retained ³⁵S-labeled mock, Id1, Id2, or Id3 protein was examined by SDS-PAGE and autoradiography. (B) 293T cells were cotransfected with GST, GST-Id1, GST-Id2, or GST-Id3 plasmid together with Flag-Mitf plasmid. After 36 hours of transfection, cell lysates were immunoprecipitated with Flag-Mitf immobilized on M2-sepharose beads. The beads were washed, resolved by SDS-PAGE, and detected by Western blotting using anti-Flag antibody (top panel). Whole-cell extracts (WCE) were also subjected directly to Western blot (WB) analysis with same antibody (bottom panel). (C) Full-length murine Mitf cDNA was prepared from TNT rabbit reticulocyte lysate as described in “Methods and materials.” Oligonucleotides spanning E-box 2 in the murine OSCAR promoter was used as probe for EMSA. Specific binding was determined by cold competition using unlabeled wild- or mutant-type probe at 10- and 100-fold molar excess concentrations (lanes 3-6). Mitf lysate and probe were incubated with indicated amounts (1-3 μg) of GST, GST-Id1, GST-Id2, or GST-Id3 (lanes 7-14). Results are representative of at least 2 independent sets of similar experiments. (D) 293T cells were cotransfected with 0.1 μg OSCAR 1.7-kb promoter luciferase reporter and 0.25 μg Mitf plasmid together with indicated amounts (0.2-2 μg) of plasmid expressing Id1, Id2, or Id3. Each well was also cotransfected with 20 ng β-galactosidase expression vector to control for transfection efficiency. Luciferase activities were normalized to β-galactosidase activity as expressed by the cotransfected plasmid. The level of 1.7-kb OSCAR reporter construct in the presence of empty expression vector was set to 1. Data represent the mean and the SE of triplicate samples. Results are representative of at least 3 independent sets of similar experiments. (E-G) BMMs were transduced with control (pMX-IRES-EGFP) or Id2 retroviruses and cultured for the indicated days with M-CSF and TRANCE. Northern blot analysis (E), RT-PCR (F), and Western blot analysis (G) were performed to assess the expression of the indicated genes.