Munc18c phosphorylated by CIRK no longer binds VAMP2. 1 μg of GST or GST-VAMP2 immobilised on glutathione-Sepharose were incubated with 1 μg of Munc18c (which had been previously treated with or without 1.8 μg CIRK for 150min) in binding buffer overnight at 4°C. Sepharose beads were washed prior to immunoblot analysis with anti-Munc18c (upper panel) or anti-phosphotyrosine antibodies (lower panel) as labelled. In the case of the anti-phosphotyrosine immunoblot, the indicated amounts of input were also loaded to reveal the presence or absence of any tyrosine phosphorylated Munc18c in the GST pull-down. Data shown are from representative experiments, repeated three times with similar results.