Gcn5 can inhibit Hda1-Tup1 associations at some promoters. (A) Sequential ChIP was used to observe Hda1-Tup1 physical interactions at specific promoters. WT, hda1 Δ and gcn5 Δ hda1 Δ cells expressing combinations of GAL
-HDA1-HA and CUP1
-TUP1-GST were induced using 4% galactose for 5 hours and 0.4 mM CuSO4 for 3 hours. ChIP reactions were first performed with antibodies against HA. Bound proteins were eluted from beads using 10 mM DTT for 30 minutes at 37°C. The eluted proteins were then incubated with anti-GST antibodies. The immune complexes were isolated again, cross links were reversed, and "end point" PCR was performed using the recovered DNA as template. (B) Two independent experiments were performed and processed using ImageJ. The means and standard errors are shown. (C) Westerns showing expression of the proteins used in the sequential ChIP experiment.