Fig. 5. Sperm stimulates protein-tyrosine phosphorylation in LD-DIM fractions in vitro. Aliquots (50 µl) of LD-DIM fraction (lanes 1-4) or non LD-DIM fraction (lanes 7 and 8) from unfertilized eggs were preincubated in the absence or the presence of jelly water-treated sperm (10⁸ sperm/ml) and subjected to protein kinase assay with or without 5 mM MgCl₂ and 1 mM ATP as described in Materials and Methods. As a control, sperm alone was also examined for its kinase activity (lanes 5 and 6). Phosphorylated proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine antibody (PY99, 1 µg/ml) without (A) or with (B) 10 mM L-phosphotyrosine. Black asterisks indicate the positions of sperm-dependent phosphorylation bands. A white asterisk indicates the position of a band that can be seen in the presence of sperm alone.