Biochemical Analysis of Histone Succinylation

Figure 3

In vitro histone acylation assays using strong cation exchange column-fractionated HepG2 nuclear extracts. (a) Schematic diagram of the fractionation of HepG2 nuclear extracts (NEs). HepG2 NEs were loaded on the HiTrap SP column and eluted with BC buffers containing the indicated KCl concentration. Each eluted protein was used for the in vitro acylation reaction. (b) Validation of HepG2 cell nuclear extracts fractionation by strong cation exchange column. Nuclear extract (NE) input and each eluted fraction (0.1, 0.3, 0.6, and 1.0 M KCl) were assessed by western blotting using anti-p300 and anti-CBP as first antibodies. (c) ¹⁴C-labeled coenzyme A incorporation into histone proteins was assessed by autoradiography. The amounts of added substrate (calf thymus histone proteins) in each reaction were visualized by Coomassie Brilliant Blue (CBB). Molecular weight is indicated on the left side of the figure, and each histone position (histones H3, H2A/B, and H4) is indicated in the right side of the figure. (d) The effect of heat inactivation was assessed. The SP 1.0 M fraction of nuclear extracts was heat-inactivated at 96°C for 10 min. The amounts of added substrate (calf thymus histone proteins) in each reaction were visualized by Coomassie Brilliant Blue (CBB). Molecular weight is indicated on the left side of the figure, and each histone position (histones H3, H2A/B, and H4) is indicated in the right side of the figure. SP: HiTrap SP column.

(a)

(b)

(c)

(d)