Deletion of HDA1 results in increased Gcn5 at promoters. (A) Steady-state Gcn5-TAP in different mutant backgrounds was determined in early log phase asynchronous cells grown at 30°C by Western blotting. Westerns were performed using antibody against TAP and the membrane was stained with Ponceau S to confirm equal protein load. (B) Plasmid borne HA-tagged GCN5 and ELP3, driven by the galactose inducible promoter, were expressed in cells lacking the proteasome ubiquitin receptor Rpn10. Cells were grown overnight in 2% glucose to early log phase. The glucose-supplemented media was washed away and the cells were resuspended in 2% galactose-supplemented media. The cells were then split with one half incubated at 37°C and the other half left at 30°C. The cells were incubated for an additional 4 hours, afterwhich proteins were harvested and analyzed with antibodies against HA or GAPDH as a load control. Controls for the experiment included endogenous APC5-TAP in rpn10 Δ cells, as well as the detection of endogenous Clb2 and Ub in WT and rpn10 Δ cells using commercially available antibodies. (C) Protein/DNA complexes were recovered from the mutants shown following GAL-induction using antibodies against either the HA epitope, total H3, or H3K9/14Ac. A mock treatment was conducted where antibody was omitted. Recovered DNA was used as template in "end point" PCR reactions using primers that amplified the promoter regions indicated. 10 μl of each reaction was separated by agarose gel electrophoresis and scanned. (D) Two independent experiments were performed. The gels were scanned and quantified using ImageJ. The means and standard errors were plotted.