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Fig. 4. H2A_T120 phosphorylation in association with H2A_K119 ubiquitylation in Hr6b-knockout pachytene spermatocytes. Triple immunostaining of wild-type (A) and Hr6b-knockout (B) early (eP), mid (mP) and late (lP) spread pachytene nuclei with anti-H2A_T120ph (blue), anti-H2A_K119ub1 (green) and anti-SYCP3 (red). The left panels show the merge of the H2A_T120ph signal and SYCP3 signal, and right panels show the merge of the H2A_K119ub1 (green) signal and SYCP3 signal (red). The arrowheads indicate the XY body. Bar, 10 µm. (C) Western blot analysis of H2A_T120ph in basic nuclear protein extracts from total testis (C). Specificity of the antibody reaction is shown by competition of the signal with the phosphorylated H2A peptide (+p) but not with the nonphosphorylated peptide (–p). The identities of protein bands are indicated. Equal amounts of protein were present in each lane, as verified by Ponceau S staining of the blot (not shown). (D) Western blot analyses of H2A_T120ph and ubiquitylated histones (ubi-his) in basic nuclear protein extracts from spermatocytes (spc) and spermatids (spt) isolated from wild-type (wt) and Hr6b-knockout (ko) mice. Asterisk indicates a non-specific protein band enriched in germ cell extracts compared with total testis extracts; the localization of H2A_{K119ub1T120ph} was verified using the localization of ubiquitylated histones visible on the same blot that was stripped and reprobed with anti-ubiquitin antibody as shown. Equal amounts of protein were present in each lane, as verified by Ponceau S staining of the blot (not shown).

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