FIGURE 4.

Munc18-1-WT but not Munc18-1-F115E or Munc18-1-F115E/E132A binds to the SNARE complex. A, assembled SNARE complex (comprising Sx1a-His, GST-SNAP25, and VAMP2) was isolated on Co²⁺ affinity beads, incubated with Munc18-1-WT or Munc18-1-F115E for 2 h at 4 °C. Following extensive washing, beads were analyzed for bound proteins using Coomassie-stained SDS-PAGE. Munc18-1-WT and Munc18-1-F115E binding to the assembled SNARE complex was confirmed by analyzing both boiled (95 °C for 5 min, denatured complex) and not boiled (SDS-resistant complex) samples. B, Western blot of the SNARE complex-mediated pulldown of Munc18-1-WT and Munc18-1-F115E/E132A probed with anti-Munc18-1 antibody. C, the amount Munc18-1-WT and Munc18-1-F115E/E132A binding to the SNARE complex on beads was averaged and expressed as percentage of Munc18-1-WT binding (mean values ± S.E., n = 4).