Phosphoamino acid analysis of ³²P-labeled enolase and the kinase activity induced by botrocetin-vWF. After in vitro kinase reactions described in Fig 9, the samples were applied on 8% SDS-PAGE. The ³²P-labeled proteins corresponding to phosphorylated enolase and 60-kD protein were excised from polyacrylamide gels. Phosphoamino acid analysis was analyzed by two-dimensional electrophoresis described in the Materials and Methods. The positions of the phosphoamino acids were determined by ninhydrin staining of standards added to each extract. P-Y, phosphotyrosine; P-T, phosphothreonine; P-S, phosphoserine. (A) Enolase; (B) 60-kD band. The data are representative of two experiments.