Fig. 5. Cytosolic NSF binds to egg membranes and is inhibited by NSF antibodies. (A) CVs were incubated with cytosol, and with and without (±) nucleotides for 1 hour at room temperature followed by washing in IM ± appropriate nucleotides. Lanes 1, 2, CVs alone; lanes 3, 4, CVs incubated with cytosol without added nucleotide; lanes 5, 6, CVs incubated with cytosol supplemented with 200 µM ATP--S; lanes 7, 8, CVs incubated with cytosol supplemented with 2 mM ATP. (B) IPCs were incubated at room temperature with cytosol at a concentration of 2 mg/ml supplemented with 200 µM ATP--S. NSF antibody was included at concentrations from 0 to 40 µg/ml as indicated. After 1 hour, dishes were extensively rinsed with IM plus 200 µM ATP--S and IPCs dissolved in sample buffer. NSF binding was visualized by western blotting.