Figure 5. SUMOylation is indispensable for transcription repression of Zbtb14.

(A) Western blot analysis (anti-HA) of HA-tagged wild type (WT), Zbtb14^K40R, and Zbtb14^K259R mutant proteins expressed in HEK293T cells in the absence or presence with the SUMO (Small Ubiquitin-like MOdifier) conjugating enzyme UBC9 and SUMO1. (B) HA-tagged WT or Zbtb14^K40R mutant protein was immunoprecipitated with an anti-HA antibody from HEK293T cells co-expressing GFP-SUMO1, and SUMOylated Zbtb14 protein was detected by western blot with an anti-GFP antibody. (C) Immunofluorescence analysis of WT (left panel) and Zbtb14^K40R mutant protein (right panel). (D) The structure of variant forms of Zbtb14, including WT, Zbtb14^K40R, and SUMO1-Zbtb14^K40R mutants (top panel). Repression of luciferase expression from the zebrafish pu.1 promoter (–0.6 kb) by Zbtb14 mutants (bottom panel). Bars showed the relative luciferase activity on the zebrafish pu.1 promoter (–0.6 kb). (Student’s t test, N=3. Error bars represent mean ± standard error of the mean (SEM). ns: not statistically significant, ***p<0.001.) (E) mRNA rescue assays in zbtb14^-/- mutant larvae. mfap4 probe was used in whole-mount in situ hybridization (WISH) to examine rescue effects of injections of zbtb14, zbtb14^K40R, and SUMO1-zbtb14^K40R mRNA. (F) Statistical result for E. The statistical significance was calculated by using one-way analysis of variance (ANOVA). The asterisk indicates a statistical difference. (N=3, 10–17 embryos were used for each experiment. Each dot represents the mean value of one experiment. Error bars represent mean ± SEM. ns: not statistically significant, ****p<0.0001.)