Fig. 6: DKC1 is SUMOylated at K413, K448, K467.

A Illustration for deletion mutant vectors encoding Flag-tagged DKC1 truncates lacking specific sequences. B Determination of SUMOylation among DKC1 deletion mutants. Following lysis of cells carrying the indicated deletion mutant encoding constructs with HA-SUMO3 or not, levels of SUMOylated DKC1 species in each were revealed by pulling down with anti-Flag gels and immunoblot probing for HA among the immunoprecipitated proteins. The expression of DKC1 and SUMO3 by transfectants (INPUT) in these studies was also confirmed by immunoblot analysis. C Immunoblot analysis of UBC9-mediated SUMOylation of wild-type and mutant DKC1 molecules. HEK293T cell lines were transfected with a normal Flag-tagged DKC1 construct, or one of constructs encoding a predicted SUMOylation-resistant mutant in which one lysine residue was replaced by arginine (K-R). These cell lines also received expression vectors encoding Myc-UBC9 and HA-SUMO molecules. Negative controls did not receive UBC9 or expressed SUMO alone. Labeled DKC1 proteins were pulled down from cell lysates with anti-Flag gels, and SUMOylated species were visualized for HA by immunoblotting. D HEK293T cell lines were transfected with Flag-labelled wild-type or mutant DKC1 (K413/448 R, K413/467 R, K448/467 R, all three sites mutated by K-R). Encoding vectors of Myc-UBC9 and HA-SUMO3 were also transfected into these cell lines. Negative control did not receive UBC9. Tagged DKC1 proteins were purified with anti-Flag gels, and the SUMOylation degree was illustrated by immunoblot assay with anti-HA antibodies after resolution by SDS-PAGE. HC, high chains of antibodies used for IP. LC, low chains of antibodies used for IP.