Fig 4

(A) Luciferase reporter assay. Bars showed the relative luciferase activity on the zebrafish c/ebpα promoter (-2.0 kb), c/ebp1 promoter (-2.1 kb), and pu.1 promoter (-1.7 kb) (Student t test, N = 3. Error bars represent mean ± SEM. **P < 0.01, ***P < 0.001). (B) Western blot analysis (anti-FLAG and anti-HA) of FLAG-Irf2bp2a, FLAG-Irf2bp2a RM, and HA-Gfi1aa expressed in HEK293 cells. The proteasome inhibitor MG132 (2.5 μM) was used to inhibit the degradation of ubiquitinated proteins. Equal protein amounts for each sample were loaded (anti-Actin). (C) HA-Gfi1aa protein was immunoprecipitated (IP) with an anti-HA antibody from HEK293 cells co-expressing Ubiquitin and FLAG-Irf2bp2a. Many more adducts of Ubiquitin were detected by western blot with an anti-Ubiquitin antibody. (D) WISH assay of mpx in WT, irf2bp2a^-/- mutant embryos, wild type and irf2bp2a^-/- mutant embryos injected with gfi1aa MO, gfi1aa^-/- embryos, and gfi1aa^-/- embryos injected with irf2bp2a MO. (E) Statistic result for D. The statistical significance was calculated by using one-way ANOVA. The asterisk indicates a statistical difference (N = 5, 19–30 embryos were used for each experiment. Each dot represents the mean value of one experiment. Error bars represent mean ± SEM. **P < 0.01; ***P < 0.001).