Luciferase assay
HEK-293T cells (3 x 104) were co-transfected, using
Lipofectamine 2000, with WT or mutant (D451G, K454fs11* or T536I) ACT1
plasmids along with NF-κB–dependent firefly luciferase plasmids pGL4.32
(Promega) and a Renilla luciferase reporter pRLTK plasmid as control of
transfection efficiency. After 24 hours, cells were stimulated with 100
ng of IL-17A (R&D) for 24 hours or left unstimulated. Luciferase was
then detected using the Dual-Glo Stop & Glo system (Promega).