The ubiquitin-proteasome pathway is involved in the downregulation of STAT1 in ALK+ ALCL cells. (A) SupM2 and Karpas 299 cells were treated with various doses of MG132 for 5 hours. pSTAT1 and STAT1 were upregulated in response to MG132 in a dose-dependent manner. (B) A time course experiment in which SupM2 and Karpas 299 cells were treated with 5 μM MG132 was performed; pSTAT1 and STAT1 were upregulated in a time-dependent manner. (C) Co-immunoprecipitation (IP) experiment using anti-STAT1 for the pull-down showed that ubiquitinated STAT1 in SupM2 cells treated with MG132 increased in a time-dependent manner (left panel). The input for this experiment is shown in the right panel. (D) SupM2 cells were transfected with the pRK5-HA(hemagglutinin)-ubiquitin (Ub) wild-type plasmid. Reciprocal pull-down experiment using anti-HA was performed, and the results also suggested that the amount of ubiquitinated STAT1 increased with MG132 treatment in a time-dependent manner. (E) SupM2 cells were treated with or without MG132 in the presence of cycloheximide (CHX). (F) Similar results were obtained with Karpas 299, and the results from both cell lines are graphically illustrated. The half-lives of STAT1 in SupM2 and Karpas 299 are 1.5 hours and 3 hours, respectively. MG132 potently extended the half-life of STAT1 in the 2 cell lines. Image J software was used to analyze the densitometry value of western blots bands.