Effects of Ub on induction of IL-6-responsive genes.

(A) KT-3 cells were serum- and rhIL-6–deprived for 12 hours and were cultured with 10 ng/mL rhIL-6 for the time indicated. To examine the effects of Ub, the cells were pretreated with 100 μg/mL Ub for the last 3 hours of the starvation period and cultured with Ub during the test period. Induction of IL-6–responsive genes was examined by Northern blot analysis. (B) Effects of Ub on IL-6–induced tyrosine phosphorylation of STAT3 and MAPK activation. KT-3 cells were cultured and treated with rhIL-6 as described above. To detect tyrosine phosphorylation of STAT3, STAT3 was immunoprecipitated from total cellular lysates with anti-STAT3 antibody and separated by SDS-PAGE, and the blot was probed with anti-phosphotyrosine mAb, 4G10. Then the filter was stripped and reprobed with anti-STAT3 antibody. MAPK activation was examined by Western blot analysis on total cellular lysates with anti-phospho MAPK antibody, which primarily recognizes activated MAPK. The filter was then reprobed with anti-MAPK antibody.