Fig. 5. Thr153 and active p38α are required for efficient transcription activation by Usf-1. (A) The Tyrosinase promoter-luciferase reporter (300 ng) was transfected into B16 melanoma cells either alone or together with the MLK and Usf-1 expression vectors (50 ng) in the indicated combinations and luciferase activity determined. (B) A GAL UAS-luciferase reporter (200 ng) was transfected into COS7 cells either alone or together with vectors expressing WT or mutant Usf-1 fused to the Gal4 DNA-binding domain in the presence or absence of co-expressed p38α/MKK6b(E) kinases as indicated. (C) Western blot of the indicated Gal4–Usf-1 chimeric proteins expressed in the transfected cells using anti-Gal4 DNA-binding domain antibody (Clontech). (D) Chromatin immunoprecipitation assay at the Tyrosinase promoter using the indicated antibodies before or after UV irradiation (254 nm, 40 J/m²) of B16 melanoma cells.