The mouse anti phosphotyrosine anti body PY99 was purchased from Santa Cruz. Rabbit antibodies against phosphorylated human MAPK, human cyclin B2, and Xenopus Mos were from BioLabs or Santa Cruz Biotechnology. Rabbit antibody against selleck bio the 85 kDa subunit of human PI 3 kinase was obtained from Upstate Cell Signaling Solution. Rabbit antibodies against human Akt, and the phosphor ylated Thr308 or phosphorylated Ser473 form of human Akt were obtained from Cell Signaling Technology. Human embryonic kidney 293 cells were grown in Dulbeccos Modified Eagles Medium supplemented with 10% fetal calf serum at 37 C/5% CO2 in a humidified incubator. A Src specific inhibitor, PP2, its inactive ana log, PP3, a PLC specific inhibitor, U73122, and a PI 3 kinase specific inhibitor, LY294002, were purchased from Calbiochem.

A23187 and bovine spleen cathepsin B were from Sigma. Leupeptin was from Peptide Institute. H2O2 was from Santoku Chem ical Industries. meth anesulfonyl fluoride hydrochloride was from Calbiochem. A synthetic tyrosine kinase sub strate peptide was synthesized and puri fied as described previously. Inhibitors,Modulators,Libraries The fluorescent Ca2 indicator fura 2 was obtained from Calbiochem. Xenopus Src was partially or fully purified from immature oocytes as described previously, and used for kinase assays in vitro. ATP was obtained from ICN. A potent PTEN inhibitor bp was purchased from Calbi ochem. Phosphatidylinositol was obtained from Ser dary Research Laboratories. PI 4 phosphate and PI 4,5 bisphosphate were prepared from bovine brain as described previously. PI 3,4,5 tri sphosphate was prepared as described.

All the phospholipids were lyophilized from the chloroform solution and suspended in distilled water or 20 mM Tris HCl by sonica tion. Protein A Sepharose was obtained from GE Health care Biosciences. Unless otherwise indicated, other chemicals were purchased from Sigma, Wakenyaku, Wako, or Nacalai Tesque. Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries Eggs, sperm, Inhibitors,Modulators,Libraries and embryos The collection of unfertilized eggs and sperm, removal of the jelly layer from eggs, and jelly water treatment of sperm were carried out as described previously. The activation of jelly layer free eggs was done by insemi nation with jelly water treated sperm or Inhibitors,Modulators,Libraries by parthenogenetic activation with the calcium iono phore A23187, H2O2, cathepsin B, or bp for the periods specified in the text.

After the activation treatments, egg samples were washed with ice cold DeBoers solution containing 110 mM NaCl, 1. 3 mM KCl, and 0. 44 mM CaCl2, pH 7. 2, immediately frozen in liquid nitrogen, and kept at 80 C. Activation was monitored by the occurrence of cortical contraction and first selleck chemical cell cleavage in a group of control eggs. Microinjection To determine the requirement of egg PI 3 kinase activity for Xenopus egg activation, the unfertilized eggs were microinjected with LY294002 as described previously.