Figure 4.
Detection of Kcr in histones by Western blotting. (A) Specificity of pan anti-Kcr antibody demonstrated by dot-spot assay using five peptide libraries with indicated amount (ng). Each peptide library contains 13 residues CXXXXXKXXXXXX, where X is a mixture of 19 amino acids (excluding cysteine), C is cysteine, and the 7th residue is a fixed lysine residue: unmodified lysine (K), Kac, propionyllysine (Kpr), butyryllysine (Kbu), and Kcr. (B) Detection of Kcr in histones. Western blotting was carried out using the histones from HeLa cells with competition of a peptide library bearing a fixed unmodified lysine (K) or Kcr. (C) Dynamics of histone Kcr in response to crotonate. The histone proteins extracted from human prostate cancer cell line Du145 incubated with 0, 50 or 100 mM crotonate for 24 hours, were Western blotted with anti-Kcr pan antibody. (D) MS/MS spectrum of PEPA KD4-crSAPAPK identified from D4-crotonate-labeled sample. The mixture of D4-, D3- and D2-crotonyl groups was used for the identification of D4-crotonyl peptide. (E) Kcr signals in core histones of S. cerevisiae, C. elegans, D. melanogaster (S2), M. musculus (MEF), as well as H. sapiens (HeLa) cells by Western blotting analysis with competition.