FIGURE 2.

PKCδ down-regulates TCR-induced activation of NF-κB. A, PKCδ dose-dependently inhibits CARMA1-induced NF-κB activation in reporter assays. HEK293 cells were transfected with the indicated mammalian expression plasmids and an NF-κB reporter plasmid for 18 h, luciferase assays were performed 16 h after transfection. B, effects of PKCδ-siRNAs on the expression of PKCδ. The PKCδ-siRNA or control siRNA (green fluorescent protein- siRNA) plasmids, together with Flag-PKCδ and Flag-MSI constructs, were transfected into HEK293 cells. Twenty-four hours after transfection, cell lysates were analyzed by immunoblotting with an anti-Flag antibody. The PKCδ bands from three independent experiments were quantitated using the Bio-Rad Quantity One Program and normalized by levels of the control protein MSI. The average levels of PKCδ are shown at the bottom of the blot. **, p < 0.01, n = 3. C, knockdown of PKCδ potentiates TCR-induced activation of NF-κB. Jurkat T cells stably transfected with PKCδ-siRNA #3 or control siRNA were stimulated by CD3/CD28 crosslinking for the indicated times. The cell lysates were analyzed by immunoblotting with the indicated antibodies. D, knockdown of PKCδ enhances IL-2 secretion upon CD3/CD28 costimulation. Jurkat T cells stably transfected with PKCδ-siRNA #1, #3, or control siRNA were stimulated with medium (mock) or CD3/CD28 crosslinking for 48 h. The culture medium was collected and subjected to ELISA. The average levels of IL-2 from three independent experiments are shown as mean ± S.D., n = 3. *, p < 0.05.