Fig. 1. NSF is required for NE formation. (A) NEs were formed by incubating Xenopus laevis egg cytosol and membranes with demembranated sperm chromatin. Reactions were performed in the presence of 1.2 µM (hexamer) bacterially expressed wild-type NSF or the dominant-negative E329Q variant, or NSF buffer as control. After 90 minutes, the samples were fixed. Chromatin and membranes were stained with 4',6-diamidino-2-phenylindole (DAPI) and 3,3'-dihexyloxacarbocyanine iodide (DiOC₆), respectively, and imaged by confocal fluorescence microscopy. Bar, 5 µm. (B) The percentage of chromatin particles with closed NEs (CNE) was determined visually by light microscopy in reactions performed as in A. Shown are means from three independent experiments (n=3) with >50 particles counted in each (error bars indicate ± s.d.).