Figure 1.

Insulin stimulation increases PI-3 kinase activity in SKN-SH and SK-N-BE(2) cells. Cells treated with insulin (100 nM) for the indicated time, were lysed and immunoprecipitated with either anti-phosphotyrosine (PY20, 5 μg) (A) or anti-p85 (B). Kinase activity was measured as described under Materials and Methods by using phosphatidyl inositol as substrate, and the product PIP was resolved by TLC. Incorporation of ³²P into PIP was imaged and quantitated using a PhosphorImager. Each bar represents the quantitated average ± SE for three independent experiments normalized to the value of unstimulated SK-N-SH cells. The anti-p85 blot (B) was obtained from the protein A beads used for PI-3 kinase activity assay.