PIAS3 inhibits the transcriptional activity of MITF through blocking the DNA binding, independent of sumoylation. (A) HEK 293T cells were transfected expression vectors encoding MITF-myc and PIAS3-GFP. Whole-cell extracts were immunoprecipitated with anti-myc antibody and immunoblotted using anti-myc antibody (top). Whole-cell extracts were immunoblotted by anti-myc and anti-green fluorescent protein (GFP) antibody (middle and bottom). (B) HEK 293T cells were cotransfected with 0.8 kb c-Fos promoter luciferase reporter, MITF, with the indicated amounts of PIAS3 (WT or C334S). Each well was also cotransfected with Renilla luciferase reporter to normalize the transfection efficiency (#P < .05; ##P < .01). (C) Chromatin immunoprecipitation for mock- or PIAS3-transduced RAW264.7 cells. Immunoprecipitated chromatin was analyzed by real-time PCR for the presence of c-Fos, cathepsin K, and TRAP promoter that were normalized to the respective input levels. Error bars in all panels represent SD. #P < .01.