Fig. 4. GATE–16 interacts specifically with NSF. (A) Bovine brain cytosol (1.2 mg) was immunoprecipitated (IP) with anti-GATE–16 and anti-NSF antibodies, or with pre-immune IgGs as control. Immunoprecipitates were washed (see Materials and methods), and proteins were eluted with 2% SDS at 95°C for 2 min and analyzed by Western blots (IB) using the indicated antibodies. The right panel (cytosol) represents 150 ng of total cytosolic proteins. (B) His₆GATE–16 (200 ng) was mixed with agarose beads coupled to anti-Myc epitope monoclonal antibodies (20 μl) in the presence or absence of Myc-tagged NSF (300 ng) for 120 min at 4°C. The immunoprecipitates were immunoblotted and reacted with anti-GATE–16 and anti-NSF antibodies. (C) ATPase activity of NSF was measured in 50 μl of reaction buffer (10 mM PIPES–KOH pH 6.8, 200 mM sucrose, 150 mM MnCl₂, 1 mM ATP and 2 mM DTT) in the presence of 1.5 μg/ml His₆NSF and the indicated concentration of His₆GATE–16 (•), or in the presence of heat-inactivated His₆GATE–16 (65°C, 30 min) (▴). Samples were incubated for 2 h at 30°C and ATPase activity was determined as described (Lill et al., 1990). The dashed line indicates ATPase activity measured in the absence of NSF. The background signal from reactions incubated with NSF and ATP on ice was subtracted.