Fig. 8. Kinase activity of AmSFK1. (A) Unfertilized (U) and fertilized eggs (F) 1, 2 or 3 minutes post sperm addition, were lysed in a buffer containing Triton X-100 and total soluble protein was subjected to immunoprecipitation with the AmSFK antibodies followed by immune complex assays in the presence of unlabeled and [-³²P]ATP. Labeled proteins were separated on 12% polyacrylamide gels, which were stained, dried and exposed to x-ray film. As a mock control (M), egg lysates were precipitated with anti-IgY beads alone. Arrow indicates the Src substrate. The position of the IgG heavy chain from the secondary antibody beads used in the immunoprecipitations, which was weakly and non-specifically labeled in the assays, is indicated. (B) Representative time course (post sperm addition) of AmSFK1 immune complex kinase assay (ICKA) as assayed by labeling of the Src substrate. (C) Anti-AmSFK1 immunoblot of the inputs used in the immune complex kinase assay shown in panel B. (D) Graph representing the average relative increase in AmSFK1 activity over time as assessed by substrate labeling normalized to the zero time point (n=4). Error bars represent standard deviation; increased activity at the 60 second and later time points are statistically significant compared to that at the zero time point (P 0.001).