Extended Data Fig. 1: SAGA acetylates Ada3 at lysines 8, 14 and 182.
a, 5 μg purified Ada2/Gcn5/Ada3 subcomplex was incubated with 0.5 mM acetyl-CoA in 40 μl HAT buffer for 30 min. The reaction components were resolved on a 15% SDS-polyacrylamide gel followed by western blots with pan acetyl-lysine antibody. b-d, Analysis of the acetylation sites on Ada3 by mass spectrometry. e, Western blot analysis of Ada3 and Gcn5 in WT, Ada3-K8R, Ada3-K14R, Ada3-K182R, Ada3-K8R/K14R, Ada3-K8R/K182R, Ada3-K14R/K182R and Ada3-3KR when grown in YP + 2% glucose (YPD). f, Dot blot assay of the specificity of anti-Ada3K14ac and anti-Ada3K182ac antibodies. The Ada3 peptides containing acetylated or unacetylated lysine 14, or lysine 182 were immunoblotted with anti-Ada3K14ac and anti-Ada3K182ac antibody, respectively. Different dots represent different diluted peptides as indicated. g, Western blot analysis of cell lysates of WT, Ada3-K14R, and Ada3-K182R with anti-Ada3K14ac and anti-Ada3K182ac antibody, respectively. h, Ada3 acetylation was unaffected by FLAG tagging. i, Analysis of Ada3 acetylation in deletion mutants of different SAGA subunits by western blots. j, Silver staining of purified Wt SAGA and SAGA Ada3-3KR. k, Ada3 was acetylated by SAGA complex at lysines 8, 14 and 182 in vitro. 20 ng purified Wt SAGA and SAGA Ada3-3KR were incubated with 0.5 mM acetyl-CoA at 30 °C for 0-30 min. For Extended Data Fig. 1e-j, shown is the typical example of two biological independent experiments. For Extended Data Fig. 1k, shown is the typical example of three biological independent experiments.
