Figure 4.

2D tryptic mapping analysis of wild-type or T12 and S15 mutant Mps1 phosphorylation. FLAG-tagged Mps1 or Mps1^T12S15→AA were expressed in 293T cells by transient transfection. Forty-eight hours after transfection, FLAG-Mps1 or FLAG-Mps1^T12S15→AA were immunoprecipitated using an anti-FLAG antibody and incubated in the presence of [γ-³²P]ATP under the in vitro kinase reaction conditions before SDS-PAGE. Bands corresponding to ³²P-labeled Mps1 or Mps1^T12S15→AA were excised and subjected to 2D phosphopeptide mapping analysis as described previously (Boyle et al., 1991).