Synergistic roles of PKCα and PKCβ in IL-2 production. Impaired cytokine responses of PKCα/β double knockout T cells are not caused by a secretion defect. (A) Western blot analysis of wild type and PKCα/β double knockout cells. Western blot shown is of whole cell lysates, with 5 × 106 naïve thymocytes or peripheral CD3+ T cells per lane. Blots were stained with antibodies against PKCα, β, θ, ɛ, ζ, PKBα and Fyn (loading control). (B) Proliferative responses of PKCα/β and PKCα-deficient CD3+ T cells were analysed in comparison to wild type littermate controls. After incubation using different stimulatory conditions cells were analysed using standard procedures for thymidine incorporation. (C) IL-2 responses of peripheral CD3+ T cells that were isolated from wild type, PKCα single knockout, PKCβ single knockout and PKCα/β double knockout mice. Cells were stimulated with anti-CD3 (precoated), with or without anti-CD28, as indicated. Cytokine levels in cell culture supernatants were analysed after 48 h by Bioplex suspension array technology. (D) Analysis of secreted versus total IL-2 from wild type and PKCα/β cells. Total IL-2 was determined after 3 cycles of freezing and thawing of cultured cells. (E) IL-2 gene expression is dramatically reduced in PKCα/β double-deficient CD3+ T cells after CD3 stimulation. Wild type and mutant CD3+ T cells were stimulated with anti-CD3 for a maximum of 20 h and IL-2 mRNA expression, normalised to Gapdh, was measured via real-time PCR. Results are shown as the mean values of at least 2 independent experiments.