Fig. 5. HMGB1 is acetylated by histone acetyltransferases. (A) The indicated purified proteins were incubated with gluthatione–Sepharose-bound GST–PCAF (+), or control gluthatione–Sepharose (–), and [¹⁴C]acetyl-CoA. After incubation, the reaction products in the supernatant were separated from the beads and electrophoresed. The gel was Coomassie stained (left), dried and autoradiographed (right). (B) Native PCAF was immunoprecipitated from mouse 3T3 fibroblasts and incubated with recombinant histones H3 and H4, recombinant full-length HMGB1 and its tail-less derivative HMGB1ΔC. [¹⁴C]acetyl-CoA was added to the reaction mix in lanes 2, 4 and 6. (C) U937 cells were incubated with LPS for the indicated times, and the cell extracts assayed by western blotting with anti-acetyl H3 antibody. For comparison, the extent of H3 acetylation after incubation with TSA is also shown. The histogram shows the average and standard error of three independent experiments (the difference after LPS treatment is significant, P < 0.05).