NF-κB luciferase reporter assay

HEK293/NF-κB-luc cells were seeded in complete DMEM media in six-well plates. The cells were transfected in triplicate with 3X HA epitope-tagged TROY or TROY variants using Effectene (Qiagen). Twenty-four hours after transfection, the transfected cells were 0.1% serum starved in the absence and presence of 1 μM ruxolitinib for 16 h and then lysed in Reporter Lysis Buffer (Promega). Luciferase activity in the cell lysates was determined by luminometry using the assay system according to the manufacturer’s protocols.