Figure 2

Coro1A modulates PKCθ-mediated effector function. (A) IL-2 promoter luciferase reporter assay performed with Jurkat T cells transfected with the constitutively active mutant PKCθ A/E and wild-type or truncated Coro1A – as indicated. Transfected cells were stimulated with the calcium ionophore ionomycin overnight. The insertion in the upper left corner shows expression of recombinant Coro1A in Jurkat T cells analysed by an anti-tag immunoblot. GFP-expressing plasmid was used as an inert protein overexpression control. (B) NF-κB-dependent promoter luciferase reporter of transfected Jurkat T cells either stimulated with ionomycin or left untreated. (C) IL-2 promoter-dependent luciferase reporter of Jurkat cells stimulated with phorbol ester/ionomycin, transfected with constitutively active mutant PKCθ A/E and stimulated with ionomycin, or alternatively, transfected with constitutively active mutants of both PKCθ and Calcineurin (CaN) and/or treated by cytochalasin (Cyt) D and PKC LMWI AEB071/Sotratstaurin as indicated. (D) Cyclin D1 promoter-dependent luciferase reporter of Jurkat cells stimulated with ionomycin and transfected with constitutively active mutants of several PKC family members and with PKCθ A/E and wild-type or truncated Coro1A, respectively. The mean ± SE of three independent experiments is shown. Statistical significance was defined with p < 0.05 (Student’s t-test) and marked with one asterisk (*). A/E: constitutively active and K/R: dominant negative mutant of PKCθ; Rac1 N17: dominant-negative mutant of Rac1; Rlu - relative luciferase activity.