Figure 1. In Vitro Tat Deacetylation by Human SIRT Proteins.

(A) Scheme of Tat deacetylation assay with immunoprecipitated SIRT1–7 proteins. Expression vectors for FLAG-tagged SIRT proteins were transfected into HEK 293 cells, immunoprecipitated, and incubated with synthetic Tat (72 amino acids) carrying an N-terminal biotin label and an acetyl group at position 50 (AcTat) in the presence of NAD⁺. Immunoprecipitated material was also analyzed in a radioactive (³H) histone deacetylase assay using an H3 peptide as a substrate.

(B) WB analysis of deacetylation reactions with antibodies specific for acetylated lysine 50 in Tat (α-AcTat), with SA-HRP, or with α-FLAG antibodies.

(C) WB of Tat deacetylation by immunoprecipitated SIRT1 in the presence or absence of NAD⁺, TSA, or nicotinamide (Nic).