Distinct TCR–CD3/CD28 signaling in Treg cell lines. Treg and Tcont cell lines were stimulated with anti-CD3/CD28–coated dynal beads for the indicated time intervals. Cell lysates were analyzed by SDS-PAGE and examined by immunoblot for expression of (A) p-Akt and Akt; (B) p-MEK1/2, p-Erk1/2, and Erk1/2; and by pull-down assay for activation of (C) Ras and (D) Rap1. Expression of total Ras and Rap1 was determined by immunoblot with the specific antibodies in whole-cell lysates. Results are representative of 4 independent experiments conducted with 4 different pairs of Treg and Tcont cell lines. ON indicates overnight culture.