Fig. 3.

VAMP-8 regulates serotonin and cathepsin D release from mast cells. (A) Mast cells from VAMP-8^−/− mice (and their wild-type littermates) were incubated with [³H]serotonin overnight. The cells were then induced for secretion by IgE cross-linking by using the indicated concentrations of DNP–BSA for 30 min or by treatment with PMA/ionomycin or ionomycin alone for 15 min. The net [³H]serotonin secretion in each condition (triggered release minus control release) is shown. The data shown are means ± SEM of seven independent experiments. (B) Mast cells isolated from VAMP-8^−/− mice (and their wild-type littermates) were induced for secretion by IgE cross-linking by using different doses of DNP–BSA for 30 min. The concentrated supernatants and lysates from 8 × 10⁵ cells were analyzed by SDS/PAGE and immunoblotting by using cathepsin D antibodies. Lysates were also blotted for β-tubulin as a loading control. The shown blots are representative of at least three independent experiments. (C) The intensity of the bands corresponding to mature cathepsin D were quantitated by densitometry, and the amount of cathepsin D released after IgE cross-linking with the indicated concentrations of DNP–BSA for 30 min or after treatment of the cells with PMA/ionomycin or ionomycin alone for 15 min was expressed as a percentage of the total amount of cathepsin D present in the supernatant and cells. The data shown are means ± SEM of three independent experiments. In all experiments shown, asterisks indicate statistically significant differences between wild-type and VAMP-8-deficient mast cells (*, P < 0.05; **, P < 0.005).