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. Author manuscript; available in PMC: 2014 Dec 23.
Published in final edited form as: Nat Commun. 2014 Jun 23;5:4234. doi: 10.1038/ncomms5234

Figure 5. SUMO1 is conjugated to the non-consensus Lys 216 on CDK6 protein.

Figure 5

(a) The amino acid sequence of CDK6 reveals 18 lysine residues as indicated (red). (b) Each of the 18 Lys (K) residues was replaced with an arginine (R) through point mutagenesis. Flag-tagged CDK6 wt and K-R mt were transfected with YFP-SUMO1 in LN229 cells. The transfected cells were lysed in a denaturing and diluted in a non-denaturing buffer. The cell lysates were immunoprecipitated with a Flag antibody and examined by western blotting using the indicated antibodies (left), revealing YFP-SUMO1-Flag-CDK6 conjugates in the cells transfected with all except K216R (arrow). (c) In vitro sumoylation assay was carried out in the reaction with the indicated elements (top) and the reaction was analyzed by western blotting using a CDK6 antibody. (d) SUMO1 and control shRNA expressing LN229 cells were transfected with Flag-CDK6 wt and K-R mt. Flag immunoprecipitates (IP) were subjected to kinase assay (top panel) and, together with whole cell lysate (WCL), examined by western blotting (bottom panel).