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. 2008 Aug 18;28(20):6384–6401. doi: 10.1128/MCB.00426-08

FIG. 9.

FIG. 9.

SIRT3 deacetylates Ku70 under in vitro and in vivo assay conditions. (A) In an acetylation buffer Flag.Ku70 was incubated with PCAF. Acetylation of protein was determined by Western blotting with antiacetyllysine antibody. (B) Deacetylation of Ku70 by SIRT3 in vitro. Flag.Ku70 was acetylated in vitro with PACF, and it was precipitated with Flag M2 beads. Acetylated Flag.Ku70 was then incubated with beads containing wt or mt SIRT3 in a deacetylation buffer with or without NAD. Both wt and mt SIRT3 were immunoprecipitated from stable HeLa cells. (C) Quantification of Ku70 deacetylation by SIRT3. (D) In vivo deacetylation of Ku70 by SIRT3. Stable cells expressing wt or mt SIRT3 were induced to overexpress with Flag.Ku70 and treated with NAM (10 mM for 24 h) and/or TSA (5 μM for 6 h) as indicated. Flag.Ku70 was immunoprecipitated, and the level of acetylation was analyzed by probing with antiacetyllysine antibody. (E) Quantification of Ku70 deacetylation by SIRT3 in vivo. Values are means of three experiments.