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Fig. 2. (A,B) Reactions were performed as in Fig. 1 after addition of either 7 µg/µl affinity-purified antibodies raised against Xenopus laevis NSF, purified preimmune IgG or antibody buffer as indicated. Samples were fixed and imaged as in Fig. 1. The percentages of CNE were determined as in Fig. 1B. Bar, 5 µm. (C) Equal fractions of total egg extract, cytosol and light membranes were analyzed by western blotting with either preimmune or anti-NSF serum. Migration of molecular mass markers in kDa is indicated. (D) Xenopus laevis egg cytosol was immunodepleted with anti-xNSF or preimmune (mock) antibodies. Depletion efficiency was analyzed by western blotting as indicated with Nup107 as control. (E,F) Membranes were either incubated without ATP for 5 minutes at 19°C to inactivate membrane-bound NSF (treated) or mock treated (untreated). Reactions were performed for 90 minutes with treated or untreated membranes in combinations with either mock- or NSF-depleted cytosol as indicated. Imaging and quantification was performed as in Fig. 1A and Fig. 1B, respectively. Note that only the combination of NSF inactivation on membranes and NSF depletion from cytosol inhibits envelope formation. Bar, 5 µm. (G,H) ER formation assays with egg extracts were performed in flow chambers in the presence of 7 µg/µl purified preimmune IgG or anti-NSF antibodies, or 1.2 µM NSF_wt or NSF_E329Q for 30 minutes. Samples were carefully washed, stained with DiOC₆ and imaged by epifluorescence microscopy. ER formation was quantified by counting the number of three-way junctions per area. Results are presented as percentage of control. All results are presented as means (n=3, error bars indicate ± s.d.).

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