PIP₂ and a preincubation step are required for FL syt-regulated fusion but are not needed for C2AB-regulated fusion activity. (A–C) Fusion assays were performed as described in Fig. 1 B, with modifications. FL syt was used in all experiments shown in the left panels; C2AB was used in all experiments shown in the right panels. Assays were performed with or without PIP₂ (A), with or without 30% PE (B), and with or without a 20-min preincubation step in EGTA before the Ca²⁺ trigger (C). Other than these specific omissions, all other components remained constant. Injection of Ca²⁺ is indicated by arrows (gray arrows show Ca²⁺ injected at 0 min, and black arrows show Ca²⁺ injected at 20 min).