FIGURE 5.

USP11 stabilizes SUMOylated PML. A, HeLa cells expressing wild-type His-tagged SUMO-2 or lysine-deficient SUMO-2 and parental HeLa cells were either depleted for USP11 by infection with lentiviral knockdown constructs or treated with an untargeted knockdown construct. After depletion of USP11, cells were lysed, and His pulldown (PD) was performed to enrich SUMOylated proteins. Total lysates and SUMO-enriched fractions were analyzed by immunoblotting (IB) using an antibody against PML. Unmodified and SUMO-modified PML are indicated for the total lysate blot. B, quantification of the PML-SUMO signal in the total lysate and SUMO-enriched lanes corresponding to the His-SUMO cell lines, comparing the PML-SUMO signal in the control versus USP11-depleted cells. Error bars represent S.D. Student's t test p value is indicated. C, total lysates from A were analyzed by immunoblotting with an antibody against USP11 as a knockdown control, and Ponceau S staining was used as a loading control. SUMO-enriched fractions from A were analyzed by immunoblotting with an antibody against SART1 as a SUMO-level control, and Ponceau S staining was used as a pulldown control. Note that SART1 modified with lysine-deficient SUMO has slightly different migration behavior due to lysine-to-arginine substitutions. D, HeLa cells were depleted of USP11 by infection with five different lentiviral knockdown constructs or treated with an untargeted knockdown construct. Subsequently, cells were lysed, and total lysates were analyzed by immunoblotting using an antibody against USP11. All five viruses were found to be efficient, and a low concentration mixture of all viruses was used for all other USP11 depletion experiments to provide an effective knockdown. Note that this experiment was performed using an acrylamide gel resolved in Tris-glycine buffer, which accounts for a slightly different observed migration size of USP11.