![Fig. 5. sE-selectin induces PI3K and Akt activation in HMVECs. / HMVECs were stimulated with sE-selectin (50 nM) for 15 minutes. In the inhibition study HMVECs were pretreated with PP2 (10 μM) for 2 hours prior to stimulation with sE-selectin, and PP2 was also present during the stimulation with sE-selectin. (A) PI3K was immunoprecipitated from the cell lysate and then the kinase assay performed using PI as the substrate and [γ-32P]–labeled ATP as the donor of phosphate ions. The [γ-32P]–labeled lipids were resolved by thin layer chromatography (TLC) and autoradiographed. This is a representative blot from 3 independent experiments. sE-selectin induced a marked increase in PI phosphorylation (*PI3P) compared with nonstimulated cells. PP2-pretreated HMVECs exhibited substantial inhibition of PI3K activation, thus indicating that PI3K lies downstream of Src kinase. (B) Cell extracts were prepared and the protein content in each sample was quantitated. Samples (20 μg) were resolved by 10% SDS-PAGE and probed with mouse monoclonal antihuman phospho-Akt (*p-Akt) antibody. To verify equal loading, the blots were stripped and reprobed with rabbit anti-Akt antibody. This is a representative blot from 3 independent experiments. (C) The ratio of p-Akt to Akt band density ± SEM from 3 independent experiments. *Represents a significant difference (P < .05) between the respective groups. sE-selectin induced a marked increase in Akt phosphorylation. Pretreatment of HMVECs with PP2 significantly inhibited Akt phosphorylation.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/101/10/10.1182_blood-2002-04-1237/4/h81034300005.jpeg?Expires=1723745602&Signature=3HD649~INfgvspKLJ4p1Vowr2MyG~qfOZKST3zfBXJ-cwCZSCfUSGwYgVzqkw9-hpaLZkfAX9nvQ8de6Xr-38NDPG50GgZjQ2jmCR07aRiu4fRMKWjtUvLGxRuEe5lD90UfGnd0qhSCotGx7Gs0Kn4WhqLEZYLOcj1PtEdupwwVnx81hmFVBNvFKp~Iu1ClQBTBWbmaOzNYrMv4UVShEHCbgkQoy~M0OdG2-cw~vQ7o6AL1IlGXG-dgg9~q88Oh6~PBukYJMC1Ql0FgjwkuXeLJLXbZ6wYzWxzKIERX~iDQk9Ar47I6-tRQvJ1Hx8-hT7zsOkMIJLGxUirNu1e~VhA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
sE-selectin induces PI3K and Akt activation in HMVECs.
HMVECs were stimulated with sE-selectin (50 nM) for 15 minutes. In the inhibition study HMVECs were pretreated with PP2 (10 μM) for 2 hours prior to stimulation with sE-selectin, and PP2 was also present during the stimulation with sE-selectin. (A) PI3K was immunoprecipitated from the cell lysate and then the kinase assay performed using PI as the substrate and [γ-32P]–labeled ATP as the donor of phosphate ions. The [γ-32P]–labeled lipids were resolved by thin layer chromatography (TLC) and autoradiographed. This is a representative blot from 3 independent experiments. sE-selectin induced a marked increase in PI phosphorylation (*PI3P) compared with nonstimulated cells. PP2-pretreated HMVECs exhibited substantial inhibition of PI3K activation, thus indicating that PI3K lies downstream of Src kinase. (B) Cell extracts were prepared and the protein content in each sample was quantitated. Samples (20 μg) were resolved by 10% SDS-PAGE and probed with mouse monoclonal antihuman phospho-Akt (*p-Akt) antibody. To verify equal loading, the blots were stripped and reprobed with rabbit anti-Akt antibody. This is a representative blot from 3 independent experiments. (C) The ratio of p-Akt to Akt band density ± SEM from 3 independent experiments. *Represents a significant difference (P < .05) between the respective groups. sE-selectin induced a marked increase in Akt phosphorylation. Pretreatment of HMVECs with PP2 significantly inhibited Akt phosphorylation.
