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.