Fig. 4

Phosphorylation of synaptosomal proteins

(a) Striatal synaptosomal proteins were preincubated with ³²P-orthophosphate and phosphorylation monitored in the presence of 100 nM FK506. Synaptosomes were lysed, and the crude synaptic vesicle fraction was recovered following centrifugation as described in Materials and Methods. The ³²P-labelled synaptic vesicle proteins from untreated and 100 nM FK506-treated synaptosomes were resolved by gel electrophoresis, the gels were dried, and an autoradiogram was prepared. Immunoblot analysis of these samples probed with anti-synapsin I Ig demonstrated equivalent amounts of synapsin in each fraction. Molecular weight markers in kilodaltons are indicated. (b) ³²P-labelled synapsin I was immunoprecipitated from phosphorylated striatal synaptosomes treated with FK506 or buffer containing 0.01% ethanol (the vehicle for FK506) as described in Materials and Methods. Duplicate samples of anti-synapsin I immunoprecipitates from control and FK506-treated synaptosomes were resolved by gel electrophoresis, gels dried down, and an autoradiogram was prepared. Synapsin I is indicated by the arrow at 73–75 kDa. Molecular weight markers are indicated in kilodaltons.