Fig. 5.

In vitro regulation of histone acetylation and chromatin accessibility by Geminin. Schematic at left illustrates the assays used to test in vitro acetylation and chromatin accessibility. (A) nuclear extract (NE, 5 μg), chromatin (2.5 μg), Acetyl CoA (50 μM), and the indicated concentrations of either Gem-GST or GST (0–50 nM) were coincubated for 10 min at 37 °C and histone acetylation was assayed by immunoblotting of acid extracted histones (using panacetylated histone H3, H4 antibodies) or stained with Silver Stain Plus (Biorad). (B) Quantitation of changes in histone acetylation. Images were collected (Biorad; Gel Doc XR) and relative intensities quantitated with Quantity One software. (C) Chromatin remodeling reactions contained 50 nM of purified Gem-GST or GST and NE (5 μg), chromatin (2.5 μg), Acetyl CoA (50 μM), and increasing concentrations of DNase I. DNA was extracted and digestion assessed on a 1.5% agarose gel, imaged using the Biorad Gel Doc XR and (D) relative intensities defined with Quantity One software.