Fig. 3.

Mutation of the SUMOylation consensus site in Ago2 abolishes SUMOylation. A) Myc-Ago2 can be modified at K402 by both SUMO1 and SUMO2. N2a cells were transfected as indicated, and SUMOylated proteins enriched on GFP-trap beads followed by western blotting. For one set, after the pulldown and extensive washing, the beads were treated with the recombinant catalytic domain of the SUMO-specific protease SENP1 to further confirm the identity of the higher molecular weight species of Ago2 as Ago2-SUMO. B) Ablation of the SUMOylation consensus site surrounding K402 reduces the residual SUMOylation observed upon mutation of K402 to alanine. Cells were transfected as indicated, followed by enrichment of YFP-SUMOylated proteins on GFP-trap beads and western blotting.