Degradation of TAK1 via ubiquitination by SOCS1. Representative immunoblots with phosphor-specific antibodies for TAK1 showed no alteration in phosphorylation after treatment with IL-1β in SOCS1-overexpressing SW1353 cells. However, TAK1 expression was reduced in pBABE-SOCS1-transfected SW1353 cells when compared with pBABE-transfected cells (A). Twenty-four hours after transfection with increasing amounts of SOCS1, new protein synthesis was inhibited with 20 μg/ml cycloheximide for 8 hours. TAK1 levels decreased with transient SOCS1 transfection in a dose-dependent manner (B). The level of TAK1 ubiquitination was analyzed via immunoprecipitation with anti-TAK1 and subsequent immunoblotting with anti-ubiquitin antibodies. The amount of ubiquitinated TAK1 protein was higher by SOCS1 overexpression (C). The treatment of MG132 as a proteasome inhibitor dose- and time-dependently increased the levels of TAK1 protein in SOCS1-overexpressing SW1353 cells (D). IP, immunoprecipitation; IB, immunoblotting.