Fig. 3. Requirements within Usf-1 for stress-induced phosphorylation in vivo. (A) MLK-mediated phosphorylation of Usf-1. B16 melanoma cells were transfected with a vector expressing SV5 epitope-tagged Usf-1 (800 ng) either alone or together with 10 ng of an MLK expression vector (pCMV.MLK). Cells were pre-treated with the SB 203580 compound (10 µM) where indicated. (B) Schematic representation of the Usf-1 protein and various point and deletion mutations expressed in the co-transfection assays with MLK shown in (C) and (D). (C and D) Vectors expressing SV5 epitope-tagged WT or the indicated Usf-1 deletion or point mutants were transfected into COS7 cells either alone or together with the MLK expression vector. Cell extracts were assayed by SDS–PAGE and western blotting using the anti-SV5 antibody. Similar results were obtained in B16 melanoma cells. (E) SV5 epitope-tagged Usf-1 (pCMV.SV5-Usf-1) or the S165A or T153A mutants were expressed in B16 melanoma cells either alone or together with MKK6b(E) and p38α as indicated.