FIGURE 5.

USP11 deubiquitinates HPV-16E7 both in vivo and in vitro. A, ubiquitination and deubiquitination of HPV-16E7 in vivo. 293T cells were transfected with expression plasmids for FLAG-16E7, Myc-USP11, and HA-ubiquitin, as indicated. At 20 h after transfection, cells were treated with 20 m MG132 for 2 h. Total cell extracts were first subjected to immunoprecipitation (IP) using α-FLAG, and then immune complexes were assayed by Western blot, and α-HA was used to detect the ubiquitination levels of FLAG-16E7. mt, FLAG-USP11 cys mt. B, USP11 cys mt has lost its ability to deubiquitinate HPV-16E7 in vitro. The purified polyubiquitinated His-16E7 was incubated with the indicated amounts of purified recombinant GST-USP11 or GST-USP11 cys mt in a deubiquitination buffer at 30 °C for 1 h. Ubiquitinated His-16E7 was detected by Western blot analysis using α-ubiquitin.