Involvement of p38 MAPK in ethanol induction of ISRE transcription and Stat1 serine phosphorylation. Panel A, Huh7 cells were transfected with 0.7 μg pISRE-luc, and 22 hours later, cells were incubated for 2 hours at the indicated μM concentrations of SB203508 (a p38 inhibitor), followed by 100 mM ethanol. Cell lysates were harvested 6 hours later and luciferase results were normalized to amounts of total cellular protein. Error bars represent standard deviations. The experiment was repeated 4 times with identical results. B, Huh7 cells were treated for 2 hours in the presence of 50 μM of various MAPK inhibitors, and stimulated with 100 mM alcohol for 20 minutes. Whole cell protein extracts were blotted for the serine phosphorylated form (S727) or total form of Stat1. The experiment was repeated twice, yielding similar results. C, Huh7 cells were transfected with control vector plasmid (Vec) or a plasmid expressing a dominant negative mutant p38 protein (p38 AGF). Twenty-four hours later, cells were not treated or treated for 20 minutes with 100 mM ethanol. Levels of S727 and total Stat1 and transfected p38 proteins were determined by western blot. The figure is representative of 2 independent experiments that produced similar results.