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Rb1 regulates post-mitotic neuron apoptosis through the Kmt5b-bcl2a/caspase pathway. AO staining in the brain at 3 dpf after overexpression of bcl2a in the nervous system (A) or treated Z-VAD-FMK (B) of siblings and zrb1-KO mutants. The white dotted line outlines the cerebellum and myelencephalon. A′, B′ Quantification of AO+ cells in all groups of (A, B) (one-way ANOVA; mean ± SEM; ****P < 0.0001; n = 10). C Dot plot showing the expression levels of kmt5b in six brain regions. The gray level represents the average expression; the dot size represents the percentage of cells expressing the marker genes. D Co-immunoprecipitation (Co-IP) analyses on the interaction between zRb1 and Kmt5b. The GPF–Kmt5b and mCherry-zRb1 plasmids were injected into wild-type zebrafish. Immunoprecipitation was performed with an antibody against GPF (upper panel) and confirmed with reciprocal immunoprecipitation experiments with antibodies against mCherry (lower panel). IgG represents a control antibody used for IPs. Input lanes contain lysate equal to one fifth of the amount used for the pull-down assays. IP indicates the antibody used for immunoprecipitation. TUNEL staining in the brain at 3 dpf after injecting huc:kmt5b-egfp plasmid (E) and kmt5b MO (F) in siblings and zrb1-KO mutants. The white dotted line outlines the cerebellum and myelencephalon. Quantification of TUNEL+ cells of the hindbrain after injecting huc:kmt5b-egfp plasmid (E′) and kmt5b MO (F′) in siblings and zrb1-KO mutants (one-way ANOVA; mean ± SEM; *P < 0.05; **P < 0.01; ****P < 0.0001; n ≥ 10).
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