The R65Q mutation does not affect binding of Munc18 proteins to syntaxins, t-SNAREs, or SNARE complexes. (A) Pull-down experiments in which equivalent amounts of recombinant His-SUMO-Munc18-2^WT or Munc18-2^R65Q were bound to nickel-nitrilotriacetic acid beads (NTA-Beads) and increasing concentrations of recombinant STX11 were added to the beads. Bound fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and coomassie blue staining. Plot shows the amount of STX11 bound to the beads normalized by the amount of Munc18-2^WT or R65Q present on the beads. (B) Pull-down experiments in which recombinant His-SNAP25/STX1 (t-SNAREs) or His-Vamp2/STX1/SNAP25 (SNARE complex) were immobilized on NTA-Beads and incubated in the presence of equivalent amounts of either untagged Munc18-1^WT or Munc18-1^R65Q. Bound fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and coomassie blue staining. Plot shows the amount of Munc18-1^WT or R65Q bound to t-SNAREs or SNARE complexes normalized by the amount of STX1 present on the beads. Gels and plots are representative of 2 independent experiments. *P < .05. A.U., arbitrary units; cdV2, soluble domain of Vamp2.