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. 2006 Jun 20;103(27):10224–10229. doi: 10.1073/pnas.0603968103

Fig. 3.

Fig. 3.

SIRT3 but not SIRT5 deacetylates AceCS2. (A) Recombinant human AceCS2myc-His purified from NAM-treated E. coli was incubated with Flag-tagged SIRT3 or SIRT5 immunoprecipitated from HEK293 cells. As a control, α-Flag immunoprecipitates from pcDNAFlag-expressing HEK293 cells were used in the reactions (left lane). Deacetylation reactions were incubated in the presence or absence of NAD+. All reactions were carried out in the presence of trichostatin A. Reactions were stopped by the addition of SDS sample buffer, boiled, and analyzed by immunoblotting. Acetylated AceCS2myc-His was detected with acetyl-lysine-specific antibodies (Ac-AceCS2myc-His). Blots were stripped and probed for total levels of AceCS2 with α-myc antibodies (AceCS2myc-His). The presence of Flag-tagged SIRT3 and SIRT5 was verified by immunoblotting with α-Flag antibodies {bottom blot [Input (Sirtuins)]}. (B) Recombinant human AceCS2 purified from NAM-treated E. coli was incubated with purified recombinant sirtuins in the presence or absence of NAD+ and in the presence of trichostatin A and analyzed as described in A. Recombinant SIRT3 or SIRT5 proteins were detected by probing with α-myc-antibodies {bottom blot [Input (Sirtuins)]}. (C) SIRT3, but not a catalytically inactive mutant of SIRT3, deacetylates AceCS2 in vitro. Deacetylation assays were performed and analyzed as described above. Where indicated, NAM was included during the incubation. (D) Overexpression of SIRT3 decreases the acetylation of ectopically expressed AceCS2. COS-1 cells were cotransfected with AceCS2HA and pcDNAFlag, SIRT3Flag, or SIRT5Flag. Acetylation of immunoprecipitated AceCS2HA was analyzed by immunoblotting with antibodies to acetylated lysine (Ac-AceCS2HA). Membranes were stripped and reprobed for total AceCS2 amounts (AceCS2HA) by probing with an α-hemagglutinin antibody. The expression of SIRT3Flag and SIRT5Flag in the total cell lysate was verified by immunoblotting with α-Flag antibodies [Sirtuins (Flag)]. (E) AceCS2 and SIRT3 coimmunoprecipitate from cells. α-Flag immune complexes from HEK293 cell lines stably expressing AceCS2Flag or an empty Flag-control vector (pcDNAFlag) were analyzed for the presence of endogenous SIRT3.