Biochemical defects in r1ΔT/r2n T cells. (A-D) Activation of biochemical pathways at early time points downstream of the TCR, IL-2R, and CD28 in r1ΔT/r2n (Cre⁺) mice compared with either r1f/r2n (Cre⁻) or WT mice. Lymph node cells were stimulated for 5 minutes, and phosphorylation of Akt (A), Erk (C), and IκB (D) was measured. (B) Kinetics of calcium mobilization was assessed using cells loaded with Fluo-3. Where indicated, CD4 and CD8 subsets are distinguished by flow cytometry. The general PI3K inhibitors wortmannin (wort) or LY294002 (LY) were used where indicated. In panel C, the MEK inhibitor U0126 was used as a specificity control in Cre⁻ cells, and the phorbol ester PMA was used as a positive control to show that the Erk pathway can be activated in Cre⁺ cells. (E-F) Activation of biochemical pathways at later time points downstream of the TCR, IL-2R, and CD28. (E) Immunoblot analysis of Bcl-X_L expression and Akt phosphorylation at 18 hours after stimulation of purified T cells (CD4 and CD8). Data are representative of at least 3 experiments, except in the case of 18-hour phospho-Akt analysis (n = 2). (F) Overlays of intracellular phosphorylation of S6 at various time points are shown. Numbers indicate the percentage of cells positive for phospho-S6.