Fig. 6.

Lys266 and Lys268 of ERα are acetylated in vivo. (A) Characterization of the AcK266/268-ERα antibody. Increasing amounts (10 ng to 300 ng) of purified acetylated or unacetylated wild type or K266/268Q mutant ERαs were subjected to polyacrylamide-SDS gel electrophoresis, followed by Western blotting with either anti-ERα antibody or anti-AcK266/268-ERα antibody. (B) Schematic diagram of the in vivo ERα acetylation assay. NA = nicotinamide. (C) and (D) Analysis of ERα acetylation in 231/ERα (C) and 293T cells transiently transfected with an ERα expression vector (D). Assays were performed as outlined in (B). The immunoprecipitated material was deacetylated by the addition of recombinant SIRT1 and NAD⁺ as indicated to demonstrate specificity of the AcK266/268-ERα antibody for the acetylated form of ERα.