Skip to main content
. 2015 Jul 28;5:12521. doi: 10.1038/srep12521

Figure 8. U2AF65 competes with MBNL1 in the export of expanded FL-HTT RNA.

Figure 8

(A) SK-N-MC cells were co-transfected with FL-HTTQ82 and U2AF65 plasmids. The cytoplasmic and nuclear RNA fractions were extracted and examined by RT-PCR 48 hours post-transfection. pcDNA3.1 plasmid was used as a control. Overexpression of U2AF65 increased the export of FL-HTTQ82 RNA. Student t-test, n = 3 biological replicates. *P < 0.05, ns = no significance, versus pcDNA3.1 group. (B) SK-N-MC cells were co-transfected with FL-HTT and U2AF65 plasmids, and levels of FL-HTT protein were assessed by western blot 72 hours post-transfection. pcDNA3.1 plasmid was used as control. Overexpression of U2AF65 increased the levels of FL-HTTQ82 protein. Student’s t-test, n = 3 biological replicates. ***P < 0.001, ns = no significance, versus pcDNA3.1 group. (C) SK-N-MC cells were triple-transfected with FL-HTTQ82, GFP-MBNL1 and U2AF65 plasmids, and levels of FL-HTTQ82 protein were assessed by western blot 72 hours post-transfection. GFP and pcDNA3.1 plasmids were used as controls, respectively. Overexpression of MBNL1 reversed the effect of U2AF65 on the level of FL-HTTQ82 and vice versa. Both experiments, one-way ANOVA, n = 3 biological replicates. *P < 0.05, **P < 0.01. (D) SK-N-MC cells were transfected with GFP-MBNL1 plasmids and MBNL1 siRNA, or U2AF65 plasmid and levels of endogenous U2AF65 and MBNL1, respectively, were examined by western blot. GFP plasmid, control siRNA and pcDNA3.1 plasmid, respectively, were used as controls. Overexpression of MBNL1 had no effect on endogenous levels of U2AF65 and vice versa. Knock-down of endogenous MBNL1 had no effect of endogenous levels of U2AF65. All experiments, Student’s t-test or one-way ANOVA, n = 3 biological replicates. ns = no significance, versus GFP group, control siRNA group and pcDNA3.1 group, respectively.