Mouse igg isotype control

MII oocytes were microinjected with 10 μM purified recombinant proteins (wild type α-SNAP, mutant α-SNAP L294A, wild type NSF and mutant NSF D1EQ) diluted in PBS if necessary. Anti-α-/β-SNAP, anti-γ-SNAP, and anti-NSF antibodies (Synaptic Systems) were microinjected at the maximum possible concentration. Anti-α-/β-SNAP, anti-γ-SNAP, mouse IgG isotype control (Novus Biologicals), and rabbit IgG isotype control (Novus Biologicals) were microinjected at 1 μg/μl. All antibodies and isotype controls were prepared in PBS. Microinjection pipettes were made by pulling borosilicate-glass capillary tubing in a mechanical puller (model P-97; Sutter Instrument Co., Novato, CA). Microinjections were performed using micromanipulators (Narishige) coupled to a Olympus IX-51 inverted microscope (Olympus). All micromanipulations were carried out in 5 μl drops of MEM/HEPES. For microinjection, needles were filled with injection solutions at the indicated concentrations, and about 7–10 pl was injected into the cytoplasm of MII oocytes by pneumatic pressure using a Pico-Injector (model PLI-100, Harvard Apparatus, Holliston, MA). Injected oocytes were used in CG exocytosis experiments after at least 1 h incubation in M16 medium, in a humidified atmosphere with 5% CO₂ at 37°C. The number of oocytes used for each experiment is indicated in the figure legends.