FIG 3.

USP6 interacts with c-Jun and mediates c-Jun deubiquitination. (A) 293T cells were transfected with the indicated plasmids. The cell lysates were immunoprecipitated with an anti-Flag antibody. The lysates (TCL) and immunoprecipitates were immunoblotted. (B) 293T cells were transfected with the indicated plasmids. The cell lysates were immunoprecipitated with an anti-HA antibody. The lysates and immunoprecipitates were immunoblotted. (C) The empty vector or a plasmid expressing USP6 or USP6 C541S was cotransfected with c-Jun–HA and Flag-ubiquitin expression plasmids into 293T cells. The cells were treated with MG132 (10 μM) for 4 h, and then the cells were lysed in RIPA buffer and the cell lysates were immunoprecipitated with anti-HA antibody. The lysates and immunoprecipitates were immunoblotted with the indicated antibodies. (D) 293T cells were cotransfected with HA–c-Jun and Flag-Ub. After 4 h of MG132 treatment, c-Jun was purified using anti-HA antibody and then incubated with GST or GST-tagged recombinant USP6 or USP6-C541S. c-Jun ubiquitylation was detected by immunoblotting with anti-FLAG. All experiments were performed three times.