FIGURE 5.

USP36 promotes DGCR8 SUMOylation. A, USP36 promotes DGCR8 SUMOylation by SUMO2. H1299 cells transfected with the indicated plasmids were subjected to Ni²⁺-NTA PD under denaturing conditions, followed by IB with anti-Flag antibody to detect DGCR8 SUMOylation. The protein expression is shown in bottom. * indicates unmodified DGCR8. B, SUMOylation of DGCR8 deletion mutants. H1299 cells were transfected with the individual DGCR8 deletion mutants without or with His-SUMO2, followed by Ni²⁺-NTA PD under denaturing conditions to detect the SUMOylation of DGCR8 fragments. The protein expression is shown in the bottom. * indicates unmodified DGCR8 fragments. C, Mutating K707 to R abolishes the SUMOylation of the C-terminus fragment of DGCR8. H1299 cells were transfected with the indicated plasmids and assayed for SUMOylation by Ni²⁺-NTA PD under denaturing conditions, followed by IB. D, Mutating K259 or K426 attenuated the SUMOylation of the central fragment of DGCR8. H1299 cells were transfected with the indicated plasmids and assayed for SUMOylation by Ni²⁺-NTA PD, followed by IB. E, Characterization of SUMO acceptor sites at DGCR8. H1299 cells transfected with His-SUMO2 together with WT Flag-DGCR8 or the indicated mutant plasmids were subjected to Ni²⁺-NTA beads PD under denaturing conditions followed by IB. 3KR indicates the Flag-DGCR8^{K259R/K426R/K707R} mutant. * indicates unmodified DGCR8.