Munc18c is a substrate for CIRK in vitro. A 1 μg Munc18c was incubated with or without 370 ng CIRK in the presence of [γ-32P]-ATP. Reactions were stopped by boiling in Laemmli buffer prior to SDS-PAGE/autoradiography. Shown are data from a representative experiment. B 1 μg Munc18c was incubated with 370 ng CIRK in the presence of [γ-32P]-ATP for 30 minutes (lane 1), 120 minutes (lane 2), or 150 minutes with 1.8 μg CIRK (lane 3). Reactions were stopped as in A. Note that different exposures of the autoradiograms are shown for clarity. C Two phosphopeptides were identified by Mass Spec analysis of phosphorylated Munc18c (peptide 2 represents mis-cleavage of peptide 1).