Munc18c-Y521E does not bind to Sx4. 1 μg of GST, Sx4-GST or the Sx4 mutants immobilised on glutathione-Sepharose, were incubated with 1 μg of either wild-type Munc18c or Munc18c-Y521E in binding buffer overnight at 4°C. An example of the input Sx4 species is shown in Figure 2. A shows an anti-Munc18c immunoblot of wild-type Munc18c and Munc18c-Y521E recombinant proteins (3% of input in each case), indicating that both species are equally recognised by the anti-Munc18c antibody used. Sepharose beads were washed prior to immunoblot analysis with anti-Munc18c (panel B; 2% of input Munc18c or Munc18c-Y521E is also shown). Data from a representative experiment is shown, repeated three times with quantitatively similar results. We also repeated these experiments using increasing amounts of Munc18c (or the Y521E mutant) between 1 and 10 μg incubated with either 0.2 or 1.0 μg of Sx4-GST. We saw no binding of Munc18c-Y521E at any of these conditions (data not shown). Quantification of these experiments is shown graphically in panel C, black bars are wild-type Munc18c binding, grey bars are Munc18c-Y521E binding. Data presented as a % of wild-type Munc18c binding wild-type Sx4 (mean ± SD).