Fig. 5. Effect of SV2A and SV2C overexpression on cytosolic and vesicular [Ca²⁺]. (A) INS-1E cells were co-infected with AdCAcAq and with adenoviruses expressing the control LacZ, SV2A or SV2C. (a) Immunoblotting analysis of the overexpressed proteins using the SV2 monoclonal antibody. (b) After cytosolic aequorin reconstitution cells were perifused with KRB containing 2.5 mM glucose and stimulated, as indicated, with the same buffer supplement with 30 mM KCl. Insulin secretion was assessed by RIA in the effluent. (c) Peak of [Ca²⁺]_c after KCl or 15 mM glucose (glc) stimulation. Values represent the mean±s.e. of seven independent traces. (B) INS-1E cells were co-infected with AdCAVAMP.Aq and with LacZ, SV2A or SV2C encoding adenoviruses. (a) Western blotting of the overexpressed proteins using anti-SV2 antibody. (b) After Ca²⁺ depletion and ER.aequorin reconstitution, cells were exposed to Ca²⁺-free KRB containing 1 mM EGTA. Where indicated, EGTA was replaced with 1.5 mM CaCl₂ and the perifusion medium supplemented with 30 mM KCl. (c) Peak of [Ca²⁺]_SG. Data are the mean±s.e. of six independent experiments.