Figure 4. Sumoylation negatively regulates Ago2 turnover.

(A) Sumoylation-deficient Ago2 mutants show increased half-lives. HeLa cells transfected with HA-Ago2-wt, HA-Ago2-4KR or HA-Ago2-K402R were treated with 50 µg/mL of cycloheximide (CHX) for the indicated times. The blot shown is a representative of four independent experiments. The cumulative results of are displayed on the graph (right panel). The initial levels of wild-type Ago2, Ago2-4KR and Ago2-K402R were normalized to 100%. Means and standard deviations are indicated (n = 4). (B) Endogenous Ago2 displays enhanced stability in Ubc^−/− MEFs in which global sumoylation is impaired. The blot shown is a representative of three independent experiments. CHX treatment and quantifications were performed as in A. Western blots for tubulin, Ubc9 and unconjugated SUMO1 are shown as controls. (C) Transient knockdown of Ubc9 results in enhanced Ago2 protein levels. HeLa cells were transfected with either a control siRNA (si-scr) or a siRNA targeting Ubc9 (si-Ubc9). Ago2 relative expression to tubulin is presented in the middle graph and normalized to 100% for siRNA-scr transfected cells. Means of three independent experiments. Graph on the right shows that Ubc9 knockdown has no effect on Ago2 mRNA expression. Total RNA extracted from HeLa cells transfected with si-scr or si-Ubc9 were analyzed by qRT-PCR with primers specific for Ubc9 and Ago2 mRNA. (D) Stable knockdown of Ubc9 results in enhanced Ago2 protein levels. Human HT1080 cells transfected with a control shRNA (scr) or a shRNA against Ubc9 were puromycin selected and processed for immunobloting with anti-Ago2 and anti-SUMO1 antibodies. Ago2 relative expression was quantified. Graph represents means of three independent experiments.