An acetylation switch actively regulates TULP3 levels in zebrafish during development.A, immunoblot showing TULP3 protein levels in zebrafish embryo lysates harvested 10 h postfertilization following treatment with DMSO or 3 μM C646 for 10 h. B, representative photos of zebrafish embryos 24 h postfertilization treated with either DMSO or 3 or 5 μM C646 for 24 h. C, graph quantifying the frequency of the phenotypes observed in panel (B); n = 120, 123 or 123 for 0, 3, and 5 μM C646 groups respectively. Mean ± S.D. shown. ∗∗∗∗ denotes p < 0.0001 (t test). D, western blot showing levels of TULP in zebrafish embryos 24 h postfertilization in the absence or presence of 8 ng TULP3 morpholino. E, representative photos of zebrafish embryos 24 h postfertilization displaying normal, delayed, mild apoptosis, and severe apoptosis phenotypes. The black and red arrows indicate apoptosis in the hindbrain and forebrain, respectively. F, graph quantifying the frequency of the phenotypes observed in panel (E); n = 271, 248, 317, or 292 for control, morpholino only, morpholino + WT, or morpholino + 2Q mutant, respectively. Mean ± S.D. shown. ∗ denotes p < 0.05, ∗∗ denotes p < 0.01, and n.s. indicates nonsignificance (t test). G, levels of TULP3 protein in morpholino-injected zebrafish rescued with either wild-type TULP3 mRNA or K316Q/K389Q TULP3 mRNA. Lysates were harvested 8 h postfertilization. H, immunoblot showing protein levels of TULP3 and p300 in zebrafish embryo lysates harvested at 4, 8, 24, and 32 h postfertilization.
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