FIG.7.

PKCαA25E induces IL-2 transcription in Jurkat and primary CD3⁺ T cells in a PKCθ-dependent manner. (A) Jurkat T cells (10 × 10⁶ per sample) were electroporated with increasing doses of either HA-PKCαA25E or PKCθA148E or pEF-BOS together with reporter IL-2-luciferase (3.8 μg) and Tk-Renilla (0.2 μg) plasmids. Eighteen hours later, Jurkat T cells were stimulated or not with ionomycin. Luciferase activity was measured as described in the legend to Fig. 2B. (B) Primary CD3⁺ T cells from healthy donors were electroporated (BTX, 360 V, 10 ms) with 60 μg of HA-PKCαA25E, PKCθA148E, or pEF-1 together with IL-2-luciferase (34 μg) and Tk-Renilla (6 μg) plasmids. Eighteen hours later, CD3⁺ T cells were stimulated or not with ionomycin, and luciferase activity was measured. (C) Jurkat T cells were electroporated with 40 μg of pFRT-PKCθ (lane 3), pFRT-PKCα (lane 2), or control vector (lane 1) together with reporter IL-2-luciferase (3.6 μg) and Tk-Renilla (0.4 μg) plasmids. Forty-eight hours later, transfected Jurkat T cells were stimulated with anti-CD3 and anti-CD28 antibodies as described in the legend to Fig. 6D. Suppression of endogenous PKCθ and PKCα protein expression was confirmed by immunoblotting. (D) Jurkat T cells (10⁶ per sample) were transfected with 0.19 μg of IL-2- or RE/AP-, NF-AT/AP-1- or AP-1-luciferase reporter plasmids with 1.8 μg of HA-PKCαA25E or pEF-BOS. Eighteen hours later, transfected Jurkat T cells were pretreated with rottlerin and stimulated with ionomycin. Four hours later, luciferase activity was measured and normalized to Tk-Renilla expression.