Degradation of STAT3, p21^WAF1, and p27^Kip1 by incorporated extracellular Ub.

(A) Binding of biotinylated extracellular Ub to STAT3 in KT-3 cells. KT-3 cells were incubated with or without biotinylated Ub for 1 hour in the presence of proteasome inhibitor MG132, and then STAT3 was immunoprecipitated from the total cellular lysates. Biotinylated Ub was visualized by peroxidase-conjugated avidin with ECL detection system (left panel). The filter was stripped and reprobed with anti-STAT3 antibody (right panel). (B) Effects of MG132 on Ub-induced STAT3 degradation in KT3 cells. KT-3 cells were pretreated with 10 μmol/L MG132 for 2 hours and then cultured with or without Ub in the presence of MG132. Total cellular lysates were obtained at the times indicated and subjected to SDS-PAGE. The filters were probed with anti-STAT3 antibody. (C) Effects of Ub on protein expression levels of p21^WAF1 and p27^Kip1 in KT-3 cells. KT-3 cells were cultured with or without 100 μg/mL Ub for the times indicated. Total cellular lysates were obtained at the times indicated and subjected to SDS-PAGE. The filters were probed with anti-p21^WAF1 or anti-p27^Kip1 Ab.