Fig. 5: DKC1 can be SUMOylated mainly by SUMO3, which is reversed by SENP3.

A, B Interaction between DKC1 and UBC9. HEK293T cells were transduced with expression vector encoding indicated molecules before lysis and co-IP test. DKC1 and UBC9 levels were determined by immunoblot assay after SDS-PAGE resolution. C DKC1 is majorly modified by SUMO2/3. HA-labelled SUMO1/2/3 was co-transfected into HEK293T cells with Flag-DKC1 construct. SUMOylation of DKC1 by different modifiers was detected by immunoblot after pulling down and separating with SDS/PAGE. D Conjugating of SUMO3 to DKC1 was confirmed by IP with anti-HA gels. E HEK293T cells were transfected with indicated combination of expression constructs encoding Flag-DKC1 and HA-tagged SUMO3 molecules—either wild-type(WT) or an active form(GG) or a variant with a G-to-A mutation(GA) responsible for preventing conjunction of SUMO3 to target protein. Modification of DKC1 by different SUMO3 variants was visualized by immunoblot following pulling down and separating with SDS/PAGE. F Colocalization of DKC1 and SUMO3. Hela cells were co-transfected with expression vectors encoding Flag-DKC1 and HA-SUMO3. The relative overlap of SENP3 protein and DKC1 protein was observed by fluorescence microscopy (blue: DAPI, red: SUMO3, green: DKC1). G SUMOylation levels of DKC1 were detected in presence of SENP3. HEK293T cells were transfected with a combination of indicated constructs before lysis and pull-down with anti-Flag gels. SUMOylation of DKC1 was determined by immunoblot assay with anti-HA antibodies after SDS-PAGE resolution in denaturing condition. H Increasing amount of expression vector encoding SENP3 decreased DKC1 SUMOylation in a dose-depend manner. The extent of DKC1 SUMOylation was detected in HEK293T cells transfected with different amounts of SENP3. I Assessment of SUMOylation of DKC1 upon expression of wild-type and catalytically inactive SENP3 mutants. Given combinations of constructs encoding wild-type SENP3, the functionally deficient C532S SENP3 (MUT), Flag-DKC1, and HA-labeled SUMO3 molecules were transfected into HEK293T cell for 24 h. Cells were lysed and DKC1 were recovered by pulling down using anti-Flag gels and then detected with immunoblotting with indicated antibodies. J Determination of SUMOylation degree of DKC1 in presence of SENP1, SENP2 or SENP3. HEK293T cells were transfected with the indicated panel, cell lysates were used to detect the SUMOylation level of DKC1 as processed in E. HC, high chains of antibodies used for IP. LC, low chains of antibodies used for IP.