Fig 1: Ube2j2−/− spermatocytes display unstable recombination intermediates and fail to form crossovers(A) Double immunofluorescence labeling of spermatocyte spreads of Ube2j2+/+ and Ube2j2−/− spermatocytes with antibodies against SYCP3 (red) and MSH4 (green). MSH4, a marker for stabilizing recombination intermediates to promote crossover formation. Scale bar, 5 μm.(B) The number of MSH4 foci decreases from zygotene to early-pachytene in Ube2j2−/− spermatocytes. Columns display means ± SEM. Each dot represents the number of MSH4 foci per nuclei. For Ube2j2+/+ group, Zygonema: n = 10, early-Pachynema: n = 9, mid-Pachynema: n = 9. For Ube2j2−/− group, Zygonema: n = 11, early-Pachynema: n = 18, mid-Pachynema: n = 7. ∗∗, p < 0.01. ∗∗∗∗, p < 0.001, Student’s t tests.(C) Double immunofluorescence labeling of spermatocyte spreads of Ube2j2+/+ and Ube2j2−/− spermatocytes with antibodies against SYCP3 (red) and RNF212 (green). RNF212, a marker for crossover-fated intermediates stabilization. Scale bar, 5 μm.(D) Double immunofluorescence labeling of spermatocyte spreads of Ube2j2+/+ and Ube2j2−/− spermatocytes with antibodies against SYCP3 (red) and HEI10 (green). HEI10, a marker for recombination intermediates stabilization, promoting RNF212 and/or MSH4-MSH5 turnover from chromosomes. Scale bar, 5 μm.(E) Double immunofluorescence labeling of spermatocyte spreads of Ube2j2+/+ and Ube2j2−/− spermatocytes with antibodies against SYCP3 (red) and MLH1 (green). MLH1, a marker for crossovers. Scale bar, 5 μm.(F) The number of RNF212 foci increases at mid-pachytene in Ube2j2−/− spermatocytes. Columns display means ± SEM. Each dot represents the number of RNF212 foci per nuclei. For Ube2j2+/+ group, early-Pachynema: n = 8, mid-Pachynema: n = 6. For Ube2j2−/− group, early-Pachynema: n = 10, mid-Pachynema: n = 8. ∗∗∗, p < 0.005, Student’s t tests.(G) The number of HEI10 foci decreases at early-pachytene in Ube2j2−/− spermatocytes. Columns display means ± SEM. Each dot represents the number of HEI10 foci per nuclei. For Ube2j2+/+ group, early-Pachynema: n = 17, mid-Pachynema: n = 6. For Ube2j2−/− group, early-Pachynema: n = 15, mid-Pachynema: n = 9. ∗, p < 0.05, Student’s t tests.(H) The number of MLH1 foci decreases in Ube2j2−/− spermatocytes. Columns display means ± SEM. Each dot represents the number of MLH1 foci per nuclei. For both Ube2j2+/+ and Ube2j2−/− group, n = 11. ∗∗∗∗, p < 0.001, Student’s t tests.
Fig 2: HEI10 as a potential CDK2 substrate.CDK2/cyclin A2 kinase assay directed against GST-tagged fusion peptides of HEI10 Ser244 (lane 3) or Ser257 (lane 5) or their serine-alanine point mutants (lanes 4 and 6, respectively). Histone H1 is shown as a positive control to indicate kinase activity of CDK2/cyclin A2 (lane 1) whereas GST tag without fusion peptide is shown in lane 2 as a negative control. Panel A shows the Coomassie staining of radiolabelled proteins to indicate equal loading in each lane. Also shown is the detection of recombinant GST-HEI10 protein using anti-HEI10 antibodies (lane 8). Panel B shows the phospho-image of each peptide (lanes 1–6) and full length HEI10 protein (lane 7) and represents the amount of radiolabelling occurring following phosphorylation by CDK2/cyclin A2.
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