FIG. 5.

p38 MAPK activity is required for IL-13-induced Stat serine 727 phosphorylation. Freshly isolated human blood monocytes (5 × 10⁶/well) were treated with IL-13 (500 pM) for an hour or left untreated and harvested. SB202190 (an inhibitor of p38 MAPK activity) was added to all the samples 30 min before the addition of IL-13, and the serine phosphorylation status of both Stat1 and Stat3 was studied on Western blots with anti-phosphoserine-Stat1 (A) and anti-phosphoserine-Stat3 (B) antibodies after running 50 μg/lane of the whole-cell extracts (preparation described under Materials and Methods) on an SDS-8% PAGE gel. Arrows indicate the positions of Stat1 (91 kDa) and Stat3 (89 kDa) calculated from the migration of molecular size markers in adjacent lanes. To ensure equal loading, the blots were stripped and reprobed with Stat1 and Stat3, respectively. These blots are shown in the lower panels. SB202190-treated lysates were also checked for Stat1 and Stat3 tyrosine phosphorylation on Western blots with anti-phosphotyrosine-Stat1 and anti-phosphotyrosine-Stat3 antibodies (C) after running the same amount of protein on SDS-8% PAGE gels as was loaded in panels A and B.