PTPase inhibitors block Fyn activation at fertilization. Groups of 500 eggs were incubated with Hanks–BSA containing 0.1% DMSO or Hanks–BSA containing 0.1% DMSO and 10 μM peroxyvanadate (POV) for 30 min at 28°C. One group was then fertilized (F) and allowed to develop for 2 min, at which time all samples were homogenized in immunoprecipitation buffer. The detergent extracts were then incubated with anti-Fyn antibody or normal rabbit IgG (Control) and immunoprecipitates were incubated in kinase buffer containing [γ-³²P]ATP and the reaction products resolved by SDS–PAGE. Autophosphorylation was detected by autoradiography over 7 days of exposure, and the position of the 57-kDa Fyn protein is indicated by the arrow.

Detection of rPTPα in the zebrafish egg: Co-immunoprecipitation with Fyn. The left panel demonstrates a phosphatase assay of immunoprecipitates prepared from 1000 zebrafish zygotes (30 min postfertilization) using anti-rPTPα-35 at 1:400 dilution (●) or control rabbit serum at the same dilution (○). The anti-rPTPα-35 immunoprecipitates contained PTPase activity evident as the removal of almost all ³²PO₄ from the peptide which eluted in fractions 10 and 11. The right panel demonstrates that the anti-rPTPα-35 antibody detects a 130-kDa protein in Western blot analysis of Fyn immunoprecipitates prepared from the membrane fraction of 1500 eggs (B). This band was not seen in blots probed with control rabbit IgG (A).