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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 4. CDK6 is conjugated by SUMO1but not SUMO2/3.

Figure 4

(a) In vitro sumoylation assay was performed in a reaction with the elements as indicated (top) and examined by western blotting using a CDK6 antibody with the free and conjugated forms of CDK6 indicated (right). (b) Three cell lines were lysed in a denaturing buffer, immunoprecipitated (IP) with a CDK6 antibody with IgG used as a negative IP control and examined by western blotting using SUMO1 and CDK6 antibody. (c) LN229 cells were lysed in a denaturing buffer supplemented with or without NEM and the endogenous CDK6 protein was immunoprecipitated using a CDK6 antibody with IgG as control and tested examined by western blotting using SUMO1 and another CDK6 antibody. (d) LN229 cells transduced or not with SUMO1 and control shRNA (top) were transfected with Flag-CDK6 and myc-UBC9 (left top). Flag IP and whole cell lysate (WCL) were tested by western blotting for the interaction of Ubc9 and CDK6. (e) LN229 cells were co-transfected with the indicated vectors (top), immunoprecipitated with a CDK6 antibody and examined by western blotting for the presence of Flag-CDK6-His-SUMO1 conjugates (right). (f) LN229 cells were co-transfected with YFP-SUMO3, YFP-SUMO1 and/or Flag-CDK6 and then immunoprecipitated with a Flag antibody. Western blotting using CDK6 and GFP antibody that recognizes YFP detected SUMO1-CDK6 conjugates as indicated (right). (g) Flag immunoprecipitate from YFP-SUMO1, Flag-CDK4 and Flag-CDK6 transfected LN229 cells were examined by western blotting for the presence of Flag-CDK6-YFP-SUMO1 conjugates.