Figure 5. Sirt2 deacetylates and stabilizes PEPCK1.

(A) Sirt2 deacetylates PEPCK1. Acetylation levels of PEPCK1 expressed and purified from 293T cells and 293T cells co-expressing Sirt1 and Sirt2 were detected. (B) Sirt2 deacetylates PEPCK1. Acetylation levels of Flag-PEPCK1 expressed and IP purified from HEK293T cells and HEK293T cells co-expressing Sirt2, Sirt2^S368D and Sirt2^H187Y, respectively, were determined. (C) Sirt2 knockdown increases PEPCK1 acetylation level. Acetylation levels of PEPCK1 expressed from HEK293T cells and HEK293T cells with Sirt2 knocked down were probed by panacetyllysine antibody. Sirt2 knockdown efficiency was monitored by realtime PCR. (D) Sirt2 knockdown decreases PEPCK1 protein level. Endogenous PEPCK1 levels of HEK293T cells and HEK293T cells with Sirt2 knocked down were determined by anti-PEPCK antibody. (E) Overexpression Sirt2 decreases PEPCK1-UBR5-C binding but overexpression P300 increases the binding. Myc-PEPCK1 and UBR5-C-C/A were expressed and purified from HEK293T cells or HEK293T co-expressing either Sirt2 or P300, interaction between PEPCK1 and UBR5-C-C/A were analyzed by the amount of PEPCK1 co-immunoprecipitated with UBR5-C-C/A. (F) Sirt2 increases ubiquitination level of PEPCK1. Flag-PEPCK1, HA-Ub and Myc-Sirt2 or its catalytic mutants were co-expressed in HEK293T cells. PEPCK1 was purified by IP. Ubiquitination level of PEPCK1 was probed by anti-HA antibody. (G) Sirtinol increases the ubiquitination level of PEPCK1. Flag-PEPCK1 and HA-Ub were co-expressed in HEK293T cells maintained under different concentrations of Sirtinol. IP purified PEPCK1 ubiquitination levels were detected. (H) Sirtinol decreases steady state PEPCK protein level. HEK293T cells were cultured at different concentrations of Sirtinol. Steady state cellular PEPCK1 level as well as PEPCK1 gene transcription level was determined. See also Figure S3.