Fig. 1

Identification of TH2BS11ph modification by mass spectrometry and characterization of the TH2BS11ph antibody. a Genome-wide replacement of H2B by TH2B histone variant in mammalian spermatocytes and spermatids [12]. b Model of the TH2B-containing nucleosome highlighting the exposed serine 11 residue. c Alignment of protein sequences of TH2B from rat and mouse. H2B sequence of rat has been given for reference. The boxed region in black represents the peptide sequence used for generation of the polyclonal antibody in rabbits. d Identification of TH2BS11ph modification by LC–MS/MS technique. The y-axis indicates the relative intensity of MS/MS spectra and the x-axis indicates the mass–charge ratio. The phosphorylated serine residue of TH2B-containing peptide fragment is highlighted in red. e ELISA assay—TH2B antibody reacts with both TH2B serine 11 phospho- and non-phosphopeptides, whereas the TH2BS11ph antibody specifically reacts with the TH2B serine 11 phosphopeptide. ELISA was carried out with the Pre-bleed immune sera, Immune sera, TH2BS11ph sera and the TH2BS11ph purified antibody; white bars represent reactivity of the mentioned sera or antibody with TH2B backbone peptide, black bars represent reactivity with H2B peptide and the orange bars represent reactivity with the TH2B serine 11 phosphopeptide. f Immunoblotting of affinity purified TH2BS11ph antibody shows reactivity with only TH2B-containing nuclear lysate. Western blotting was performed with anti-TH2BS11ph antibody against liver nuclear lysates, testes nuclear lysates and recombinant TH2B (rTH2B). Coomassie stained gel is given for reference on the left. g Peptide competition assay—first lane represents no peptide control, second lane TH2BS11ph antibody pre-incubated with TH2B serine 11 phosphopeptide, third lane TH2BS11ph antibody pre-incubated with TH2B backbone peptide. Western blot was carried out with the antibodies indicated as alpha alongside the blot