CIC missense variants affected protein expression levels but not localization
To determine whether the variants affect CIC protein subcellular localization or their level of expression, functional analyses were performed on the CIC variants. As shown in Fig. 2A, wildtype GFP-CIC was primarily located in the nucleus as expected. All eight variants were also located in the nucleus, indicating that none of these variants affected the cellular localization of CIC protein. Interestingly, we noticed that some of the mutants showed a weaker nuclear staining under the same exposure conditions. We speculated that the expression level of CIC may be affected by the missense mutations. We subsequently performed western blotting assays to investigate the effect of these variants on the CIC protein levels. As shown in Figure 2B, GFP-CIC protein of all eight variants were less abundant compared to the wildtype (p<0.05), which demonstrated that the missense variants may decrease CIC protein expression or potentially affect protein stability.