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. 2013 Jul 29;110(33):13546–13551. doi: 10.1073/pnas.1301463110

Fig. 3.

Fig. 3.

SIRT2 Interacts in a complex with and deacetylates CDK9. (A and B) 293T cells were transfected with Flag-HA-CDK9 and/or SIRT2-GFP, harvested and immunoprecipitated (IPed) with either anti-Flag M2 beads (A) or anti-GFP antibody. (B) Bound proteins were washed, separated by SDS/PAGE, and immunoblotted with antibodies against GFP, Flag, and GAPDH. (C) Endogenous SIRT2 or CDK9 was immunopreciptated from HeLa cell lysates treated with or without HU. Immunocomplexes were washed, separated by SDS/PAGE, and immunoblotted with antibodies against SIRT2 and CDK9. (D) Acetylated CDK9 was isolated from 293T cells transfected with FLAG-CDK9, p300/CBP, and pCAF and incubated in an in vitro deacetylation assay with FLAG-SIRT2 isolated from 293T cells in the presence of TSA with or without NAD and nicotinamide. The reaction mixtures were separated by SDS/PAGE and immunoblotted with antibodies against acetyllysine, SIRT2, and CDK9. (E) HeLa cells were transfected with FLAG-CDK9 and HA-SIRT2 WT or HY in the presence of TSA with or without nicotinamide, IPed with an anti-FLAG antibody, separated by SDS/PAGE, and immunoblotted with antibodies against HA, acetyllysine, and FLAG. See also Fig. S3.