Pathogenic UNC13D missense variants cause reduced munc13-4 expression. HEK293T cells were transfected with FLAG-tagged cDNA carrying wild-type or missense mutated UNC13D sequences. Cells were harvested after overnight culture (A) or after additional incubation in the presence of cycloheximide for 0, 5, and 10 hours (B), and munc13-4 protein expression levels were analyzed by western blotting. (C) Relative expression of variant munc13-4 proteins at the indicated time points after treatment with cycloheximide. V1 to V15 are the UNC13D variants listed in Table 1. Results in panels A and B are representative of >3 independent experiments. Error bars indicate the mean ± SD. *P < .05; **P < .01; ***P < .001. M, mock; WT, wild type.