Figure 9.

NSF can rescue the inhibition of ISG fusion after NEM treatment. (a) Immunoblot of a PNS, membrane pellet (MP) obtained from the PNS, PC2-ISGs, and rat liver cytosol with antibodies directed against p97, p47, NSF, and α-SNAP. The blot was prepared from material loaded in amounts which are proportional to that in the assay, i.e., 110 μg of a PNS, 38.5 μg of MP, 4.1 μg of ISGs, 49 μg of rat liver cytosol. Bound antibodies were detected using ¹²⁵I-protein A. (b) NEM treatment was performed on the membrane pellet obtained from a PNS of PC12 cells labeled with [³⁵S]sulfate for 30 min or rat liver cytosol or both. A fusion assay was performed with the treated or untreated components using 10 μl of PC2-ISGs. NSF (19 μg/ml; 230 nM) or p97/ p47 (28 μg/ml p97; 290 nM p97) was added to NEM-treated membranes and cytosol before incubation for fusion. (c) NSF can rescue NEM inhibition of fusion. NEM-treated membrane pellet was incubated with either rat liver cytosol or NEM-treated rat liver cytosol in the presence or absence of NSF, p94/p47 in a standard fusion assay with 10 μl of PC2-ISGs. Values shown are after subtraction of the PC2-independent background and are representative of three independent experiments for NSF and two independent experiments for p97/47 performed in duplicates or triplicates (error bars are ±SD of the mean).