Fig. 6. Characterization of sperm-dependent PTK activity in egg LD-DIM fraction. (A) LD-DIM fraction (50 µl) was preincubated in the absence or the presence of jelly water-treated sperm (10⁸ sperm/ml). The samples were subjected to protein kinase assay in the presence of either DMSO alone (0.2%, lanes 1 and 2), 10 µM PP2 (lane 3), 10 µM PP3 (lane 4), 50 µM genistein (lane 5) or 50 µM daidzein (lane 6). (A) Phosphorylation was carried out with non-labeled ATP and analyzed by anti-phosphotyrosine immunoblotting as in Fig. 5. (B) Phosphorylation was carried out with [-³²P]ATP and analyzed by BAS2000 phosphoimaging analyzer. (C) Phosphorylation was performed as in B in the presence of Cdc2 peptide. A phosphoimage for ³²P-labeled peptide is shown. (D) LD-DIM fraction was solubilized with extraction buffer containing 2% (v/v) n-octyl-ß-D-glucoside (Wako). The samples (500 µl) were immunoprecipitated with either anti-Xyk antibody (lane 2, 1 µl of rabbit serum) or preimmune antibody (lane 3, 1 µl of rabbit serum). The immunoprecipitates were subjected to protein kinase assay and analyzed by immunoblotting with anti-pY416 antibody (top panel). We also performed protein kinase assay of the immunoprecipitates in the presence of [-³²P]ATP and Cdc2 peptide. Autophosphorylated Xyk (middle panel) and phosphorylated peptide (bottom panel) were visualized as in B. Intact LD-DIM fraction was used as a control in each assay (lane 1).

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