Fig. 2. Stress-induced phosphorylation of Usf-1. (A) Western blotting analysis using anti-Usf-1 Ab (Santa Cruz) of B16 melanoma cells, before and after UV irradiation (254 nm, 40 J/m²) or osmotic shock (0.5 M sorbitol, 20 min), at indicated time post-treatment. The 45 kDa band corresponds to the phosphorylated form of Usf-1 described previously (Galibert et al., 1997). (B) Stress-induced phosphorylation of Usf-1 is abolished when B16 melanoma cells are pre-treated with the SB 203580 compound (10 µM, 20 min prior to UVC stimulation), a highly specific inhibitor of the p38 family kinases, but not with the MEK inhibitors PD98059 (50 µM) or U0126 (10 µM) or the H89 (10 µM) inhibitor of MSK1. (C) A time course of induction of Tyrosinase expression in 501mel cells in response to UV irradiation in the presence or absence of the SB 203580 compound. Tyrosinase mRNA was detected by RT–PCR and compared with the levels of a G3PDH control. Protein extracts from the corresponding time points were western blotted and probed for Usf-1, total p38 or double-phospho-p38. Similar results have been obtained using B16 cells.