FIG 5.

USP20 deubiquitinates ERK3 in vivo and in vitro. (A) 293T cells were transfected with pcDNA3-HA-ERK3-GST, pMT123-HA-Ubiquitin, and pCMV6-XL4-USP20 as indicated. After 48 h, the cells were lysed, and ERK3 was pulled down by using glutathione-agarose beads. Ubiquitination was analyzed by immunoblotting with anti-HA antibody. (B) Cells were processed as described above for panel A, and purified HA-ERK3-GST was incubated with purified recombinant USP20 in deubiquitination buffer for 2 h. Ubiquitination of ERK3 was assessed by immunoblotting with anti-HA antibody. Ub, ubiquitin.