Figure 2: USP11 deubiquitinates PML and antagonizes the function of PML ubiquitin ligases.

(a,b) USP11 shRNAs increase the ubiquitination levels of PML-I (a) and PML-IV (b). Immunoprecipitation and/or western blot analysis of 293T cells transduced with lentivirus expressing control or USP11 shRNAs and transfected with indicated constructs. (c) Tandem affinity purification of ubiquitinated endogenous PML under denaturing conditions form U87 cells transduced with lentivirus expressing control or USP11 shRNAs and transfected with His-ubiquitin. (d) USP11 decreases PML-I ubiquitination in vivo. PML-I ubiquitination in 293T cells transfected with indicated constructs was analysed as in a. (e) In vitro deubiquitination of PML by USP11. Purified PML-I was subjected to in vitro ubiquitination reaction in the presence of E1, E2 and Roc1–Cul3–KLHL20 complex and the resulting products were incubated with baculovirally purified USP11 or its mutant for deubiquitination (Methods). The reaction products were examined by western blot with indicated antibodies.