RTA promotes K48 and K63-linked ubiquitylation of STAT6.

(A) RTA induced STAT6 ubiquitylation. HEK293T cells were co-transfected with different expressing plasmids as indicated. At 36 h post-transfection, cells were treated with proteasomal inhibitor MG132 for 6 h before harvesting and lysing for immunoprecipitation (IP) and immunoblotting (IB). (B) K48 and K63-linked ubiquitylation of exogenous STAT6 was induced by RTA. HEK293T cells were co-transfected and treated as in panel A. HA-tagged wild type (WT) ubiquitin and its lysine mutants containing only K6, K48 or K63 were used. (C) K48 and K63-linked ubiquitylation of endogenous STAT6 was significantly induced during KSHV reactivation. BCBL1 cells were treated with TPA and sodium butyrate (T/NB) for 24 h, followed by MG132 treatment for 8 h before harvesting. Whole cell lysates (WCLs) were lysed for denatured immunoprecipitation (IP) using antibodies specific against K48 or K63 polyubiquitin and immunoblotting (IB) as indicated in the figure.