Fig. 2. Anti-phospho-H3 antibodies recognize phosphorylated histone H3 but not histone H3 that is both phosphorylated and acetylated. Quiescent C3H 10T1/2 cells received no pretreatment (lanes 1–4) or were pretreated with 500 ng/ml TSA for 4 h (lanes 5–8) or 5 mM sodium butyrate for 5 h (NaBu, lanes 9–12). For each set, cells were stimulated with 50 ng/ml EGF plus 10 µg/ml anisomycin (EAn) for 30 min (lanes 2, 6 and 10), 45 min (lanes 3, 7 and 11) or 60 min (lanes 4, 8 and 12); lanes 1, 5 and 9 contain unstimulated cells. Acid-soluble nuclear proteins were resolved on 15% acid–urea gels, transferred to PVDF membrane and then analysed by western blotting using anti-acetyl-H3 antibodies (panel i), anti-phospho-H3 antibodies (panel ii) or anti-HMG-14 antibodies (panel iii). Coomassie-stained acid–urea gel is shown in panel iv. The positions of the modified forms of histone H3 are numbered corresponding to the number of post-translational modifications visualized by Coomassie staining of gels or Ponceau S staining of PVDF membrane after transfer (0 = no modification).