Fig. 3.

Increased turnover of Munc18-1 mutants in neurons. a Total protein levels of Munc18-1. WT and mutant Munc18-1b were expressed in primary neurons infected with lentiviral vectors expressing cre recombinase. Total protein levels were quantified by immunoblotting, normalized to the levels of the synaptic protein synaptophysin-1 (SypI). Data are means ± SEM (*p < 0.05, ***p < 0.001 by Student’s t test; n = 3 independent experiments). b Turnover of Munc18-1 by cycloheximide chase. Neurons as in a were subjected to a cycloheximide (CHX) chase experiment for the indicated time to stop protein translation. Remaining protein levels were quantified by immunoblotting, normalized to α-tubulin levels. Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA; n = 3 independent experiments). c–f Turnover of Munc18-1 by Dendra2 photoconversion. HEK293T cells were transfected with WT or mutant Munc18-1b:Dendra2 fusion constructs. Two days after transfection, expressed Dendra2 was photoconverted. The green signal was quantified before and after photoconversion (c, d), and the red signal was quantified at 0, 3, and 24 h after photoconversion (e, f). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test in d and f, and by two-way ANOVA in e; n = 4–6 independent experiments). Scale bar in c = 50 µm