The PIAS3_{82-132; Y94P} mutation has lost its ability both to interact with MITF and STAT3 and to inhibit their transcriptional activity. (A) In vitro-translated and [³⁵S]-labeled PIAS3_82-132, PIAS3_{82-132; Y94P}, and PIAS3_{82-132; Q125P, L126P} were incubated with GST-MITF, GST-STAT3, or GST immobilized on glutathione-sepharose beads. Retained [³⁵S]-labeled PIAS3_82-132, PIAS3_{82-132; Y94P}, and PIAS3_{82-132; Q125P, L126P} were determined by SDS-PAGE and autoradiography. One representative of 3 experiments is shown. (B) Luciferase reporter plasmids, containing a MITF promoter region of the mMCP-6, or a STAT3 reporter gene, M67, were cotransfected into NIH-3T3 cells, with expression plasmids containing PIAS3_1-628, PIAS3_82-132, PIAS3_{82-132; Y94P}, or PIAS3_{82-132; Q125P, L126P}. Luciferase activity of lysed cells was measured and normalized against protein concentration. For each transfection, the total DNA concentration was constant by complementing with the empty vector pcDNA. The mean ± SE of 4 experiments is shown. (C) Quantitative RT-PCR analysis of MITF target gene (TRP) from transfected RBL cells. Cells were nuclear transfected with expression plasmids containing PIAS3_1-628, PIAS3_82-132, PIAS3_{82-132; Y94P}, PIAS3_{82-132; Q126P, L125P}, or no insert. Transcript levels were normalized to actin and performed in triplicates. One representative of 3 experiments is shown.