Fig. 2. Soluble proteins do not reverse inhibition of CV exocytosis by thiol reagents. (A) CSCs were prepared in DTT-free IM and were treated for 1 hour at room temperature (20°C) with gentle mixing with the following. No thiol reagent (open circles), 1 mM NEM (closed circles), 200 µM D10PDP (open triangles) or 50 µM AMSDS (closed triangles). The CSC were centrifuged at 700 g for 1 minute and suspended in fresh IM before the addition of Ca²⁺ and measurement of exocytosis. Mean±standard error of the mean are shown, n=3 except for AMSDS where mean±standard deviation are shown, n=2. (B) After the treatment described in A, the CSC were washed by a further centrifugation step and were incubated with cytosol at a protein concentration of 10 mg/ml (0.1 µg NSF/mg protein) and 5 mM ATP for 30 minutes. Ca²⁺ was added and exocytosis measured. Mean±s.e.m. are shown, n=3 except for AMSDS where mean±s.d. are shown, n=2.