H4 is mainly deacetylated on K16. A) MS/MS spectrum of the mono-acetylated H4 tryptic peptide Gly4-Arg17 (m/z 768.946) from a SenRAF sample. Two rounds of propionylation were performed before and after trypsin digestion to propionylate free unmodified lysines and N-termini. Consequently, the 14 Da differences shown in y5+ and b9+ fragment ions represent the mass difference between acetylated and propionylated lysine. The spectrum is a mean of ten experiments along the chromatographic peak. Acetylation on K16 is calculated based on the ratio of the specific daughter ions y5+ and b9+ shown as insert in Prolif. and SenRAF samples. K16Ac abundance is the mean of three independent biological replicates (mean coefficient of variation, CV = 3.2%). B) Relative distribution of lysine acetylation on mono-acetylated H4 calculated from the MS/MS spectra on Gly4-Arg17 tryptic peptides. The relative abundances of H4 mono-acetylation come from Figure 1D. C) Immunoblot showing the levels of H4-K8Ac, H4-K12Ac and H4-K16Ac in acid-extracted histones. Loading control: Coomassie blue staining of histones. Prolif.: proliferating WI-38hTERT/GFP-RAF-ER; SenRAF: WI-38hTERT/GFP-RAF-ER + 20 nM 4-HT.