Fig. 1

The PKCθ-E2 replacement mutant shows impaired IL-2 transactivation in Jurkat T cells. a Schematic depiction of the domain structure of PKCθ and the wild type (wt) and mutated (mut) aa sequence of the targeted exon 2 (E2). b IL-2 promoter luciferase reporter assay performed with Jurkat T cells transfected with the constitutively active mutant PKCθ A148E bearing either the wt or mutated E2 sequence – as indicated. Transfection with the empty vector was used as a control. Transfected cells were stimulated overnight with the calcium ionophore ionomycin. c NFAT-AP1-dependent promoter luciferase reporter assay using different concentrations of the PKCθ expression plasmids as indicated in the figure was performed similar to the assay shown in (b). d NFAT-AP1-dependent promoter luciferase reporter assay performed with HEK-293T cells transfected either with empty vector, PKCθ-E2^wt or PKCθ-E2^mut. One day after transfection cells were stimulated overnight with PDBu/ionomycin. Expression of recombinant PKCθ (wt or E2^mut) and endogenous actin was analyzed by immunoblot. The bar graph summarizes results of 3 independent experiments (mean ± SEM) and immunoblots of one experiment are shown