Figure 2
Subcellular localization and tyrosine phosphorylation of an 85-kDa subunit of PI 3-kinase before and after fertilization of Xenopus eggs. (A) Triton X-100-solubilized extracts of Xenopus unfertilized eggs (lane 1, 100 μg/lane) and 293 human embryonic kidney cells (lane 2, 20 μg/lane) were separated by SDS-PAGE and analyzed by immunoblotting with the anti-p85 subunit of PI 3-kinase antibody. An asterisk indicates the position of the immunoreactive 85-kDa protein (doublets in the egg extracts). Molecular size markers (in kDa) used are also indicated. (B) Low density, detergent-insoluble membrane (LD-DIM) (left panels) and detergent-soluble (right panels) fractions were prepared from Xenopus eggs that had been untreated (lanes 1 and 6) or activated by 106/ml sperm (lanes 2 and 7), 0.5 μM A23187 (lanes 3 and 8), 10 mM H2O2 (lanes 4 and 9), or 5 U/ml cathepsin B (lanes 5 and 10). Protein samples (LD-DIM fractions, 5 μg/lane; detergent-soluble fractions, 800 μg/lane) were immunoprecipitated with the anti-p85 subunit of PI 3-kinase antibody and the immunoprecipitates were analyzed by immunoblotting with the same antibody (IB: p85) or with the anti-phosphotyrosine antibody (IB: phospho-p85). Asterisks indicate the positions of the p85 bands in each panel. (C) LD-DIM fractions were prepared from Xenopus eggs that had been inseminated for the periods indicated (0-40 min). Protein samples (5 μg/lane) were analyzed for the presence of PLCγ, p85 subunit of PI 3-kinase, or tyrosine-phosphorylated UPIII (pUPIII) by immunoprecipitation and immunoblotting (IB) as described in "Methods". Triton X-100-solubilized extracts of Xenopus unfertilized eggs (100 μg/lane) were also analyzed for the presence of phosphorylated MAPK (pMAPK) by direct immunoblotting as in Figure 1C. Asterisks indicate the positions of the protein bands of interest.