Fig. 3

Detection of Yes kinase activity in zebrafish embryos. Detergent extracts prepared from 100 embryos collected at 5.25 h postinsemination were incubated with control serum (A) or the partially purified anti-Yes antibody (B) at 1:250 dilution. Immune complexes were collected with protein A-Sepharose, washed, and incubated in a kinase reaction as described in Materials and methods. The reaction products were resolved by SDS-PAGE, treated with 1N KOH to reduce contamination by ³²P-Ser and ³²P-Thr, then the phosphoproteins were detected by autoradiography. The anti-Yes antibody immunoprecipitated kinase activity evident as a 62-kDa band (arrow). An additional protein of 110–120 kDa was also phosphorylated in these reactions. Autophosphorylation assays of immunoprecipitates prepared at different times during development demonstrated that Yes kinase activity was detected in the unfertilized egg (C), at 3.5 h (D), and 5.25 h post fertilization (E).