FIG. 3.

Wwp2 ubiquitinates Rpb1 both in vivo and in vitro. (A) Wwp2 targets CTD of Rpb1 for ubiquitination in vitro. Various purified proteins as indicated were incubated in ubiquitination buffer. Ubiquitinated CTD of Rpb1 was visualized by Western blotting with antibody against ubiquitin (left panel) and CTD of Rpb1 (right panel). (B) Wwp2 enhances ubiquitination of Rpb1 in vivo. HEK 293 cells were transfected with expression vectors as indicated. Cells were treated with or without proteasome inhibitor (MG132) before harvest. Exogenous Rpb1 was immunoprecipitated with anti-Flag antibody from the denatured cell lysate and ubiquitinated Rpb1 was analyzed by Western blotting with anti-His antibody (top panel). Wwp2 expressed in the cells was confirmed by immunoblotting with anti-Wwp2 antibody (bottom panel). (C) Wwp2 protein level regulates the ubiquitination level of endogenous Rpb1 in F9 cells. RNAi Wwp2-2 F9 cells induced with or without Tc for 3 days were treated with MG132 before harvest. Endogenous Rpb1 was immunoprecipitated with its antibodies from the denatured cell lysate, and ubiquitinated Rpb1 was analyzed by Western blotting with anti-ubiquitin antibody. Wwp2 expression levels in the cells were confirmed by immunoblotting with anti-Wwp2 antibody. Tubulin was used as loading control.