Munc18c phosphorylated by CIRK no longer binds Sx4. A 1 μg of GST, Sx4-GST or the Sx4 mutants immobilised on glutathione-Sepharose were incubated with 1 μg of Munc18c (previously treated with or without 370 ng CIRK for 150min) in binding buffer overnight at 4°C. 6% of the samples were removed and subjected to SDS-PAGE and Coomassie stained to assess inputs (upper panels). Sepharose beads were washed prior to immunoblot analysis with anti-Munc18c (10% of eluted material; lower panels). Data shown are from representative experiments, repeated three times with similar results. B Shows quantification of the data from 3 experiments of this type. Levels of control (non-phosphorylated) Munc18c bound to the different Sx4 species is expressed as a % of the level bound to wild-type Sx4 (black bars), (mean ± SD). Data from phosphorylated Munc18c (grey bars) is also shown. Note that the level of binding of phosphorylated Munc18c to wild-type Sx4 is decreased by ~30%. Quantification of the extent of phosphorylation of Munc18c under these conditions revealed that a roughly equivalent fraction of Munc18c is phosphorylated. See text for details. The amount of Munc18c captured by the different Sx4 species is in good agreement with published data . C The material eluted from the beads was also probed with anti-phosphotyrosine antibodies (40% of eluted material loaded per lane) as labelled. In this case, the indicated amounts of input were also loaded in order to confirm 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.