Fig. 5

List of key proteins associated with H1t-positive chromatin fragments in pachytene spermatocytes as determined by mass spectrometry. a Coomassie-stained gel showing the success of the H1t-ChIP technique. The first lane represents the protein marker, the second lane is the ChIP carried out using the rabbit preimmune IgG antibodies and the third lane is the ChIP carried out using H1t antibodies. b The important proteins that are associated with H1t-containing oligo nucleosomes can be broadly divided into four major classes: nucleolar function, heterochromatin and repeat-associated proteins, MIWI-associated proteins and other important proteins. The proteins indicated in red color have been selected for further validation by co-IP assays. c Validation of H1t-associated proteins by ChIP-western blotting technique. To validate further the association of key proteins with H1t-associated chromatin, we carried out western blot analysis of the H1t-positive oligo nucleosomes. We observe proteins related to the nucleolus (NPM1), heterochromatin related (Hormad1, Trim28, HP1β, HP1γ), PIWI pathway-related (PIWIL1, Hspa2) are associated with H1t-containing ChIP fraction. The first lane in all the blots represents the 10% input fraction, second lane ChIP with the non-specific isotype control, and the third lane ChIP with the H1t antibody, and the fourth lane is the ChIP carried out along with addition of C-terminal H1t recombinant protein fragment (10 µg). The antibodies labeled in alpha alongside the blot represent the antibodies used for western blotting