Fig. 3. NSF and p97 are required throughout NE formation. (A) The dominant-negative mutant p97_D2 inhibits NE formation specifically. NE formation reactions were performed in egg extract in the presence of buffer, or 12.5 µM (hexamer) of p97_D2 or the double-mutant protein p97_D2-K251A. The percentage of closed NE was determined after 75 minutes (n=3, error bars indicate ± s.d.). (B-D) Staging experiments. Three sets of NE formation reactions were performed in parallel. From the first, samples were taken, fixed and stained at indicated time points. Representative intermediates were imaged by confocal microscopy and presented as maximum intensity projections (B), and the percentage of closed NEs was determined (C). The other two sets were supplemented with the dominant-negative mutants NSF_E329Q (2.3 µM) or p97_D2 (12.5 µM), respectively, at the indicated times. The reactions were further incubated for a total of 75 minutes, then fixed and the percentage of CNE determined (D). Note that the reactions remained sensitive to addition of the mutants until completion of NE formation. Results are presented as means (n=3, error bars indicate ± s.d.).