Figure 6. Human ZBTB14S8F mutant is a loss-of-function transcription factor.
(A) 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 and ZBTB14S8F mRNA. (B) Statistical result for A. The statistical significance was calculated by using one-way analysis of variance (ANOVA). The asterisk indicates a statistical difference. (N=3, 10–16 embryos were used for each experiment. Each dot represents the mean value of one experiment. Error bars represent mean ± standard error of the mean (SEM). ns: not statistically significant, ****p<0.0001.) (C) Quantitative reverse transcriptase polymerase chain reaction analysis of ZBTB14 and ZBTB14S8F transfected in HEK293T cells. To determine the relative expression rate, data were normalized to the expression level of wild type (WT) groups (which were set to 1.0) after normalized to the internal control of β-actin. (Student’s t test, N=3. Error bars represent mean ± SEM. ns: not statistically significant.) (D) Western blot analysis (anti-HA) of HA-tagged WT, ZBTB14S8A, ZBTB14S8D, ZBTB14S8F, ZBTB14S8W, and ZBTB14S8Y mutant proteins expressed in HEK293T cells in the absence or presence with baf A1 or MG132. GAPDH served as internal control.