Luc2p stat5 re hygro

The HEK293T cell line (American Type Culture Collection) was maintained in DMEM (Invitrogen) containing 10% FBS, 2 mM glutamine, and penicillin and streptomycin (100 U/mL each; Invitrogen). HEK293T cells were transfected using Turbofect (Thermo Scientific). Luciferase reporter assays used a previously published method (31 (link)). Cells were transfected with 1 μg of the reporter plasmid, pGL4.52 [luc2P/STAT5 RE/Hygro] (Promega Corp.), 25 ng of either human WT STAT5B or Q206R STAT5B expression plasmid (pCI-STAT5B), 2 μg of pME18S-IL-2RB, 500 ng of pME18S-IL-2RG, 250 ng of pME18S-JAK3, and 0.1 ng of the transfection control reporter plasmid, pRL-CMV Luciferase (Promega Corp.). For dual luciferase assay, 30 ng of 3xFLAG tagged STAT5B construct (p3xF-STAT5B) and a variable amount of HA-tagged Q206R (pHA-STAT5B Q206R) with a normalizing amount of empty vector (pCI-HA) were mixed with the prepared cocktail for transfection using Turbofect (Thermofisher) following the manufacturer’s instructions. Twenty-four hours after transfection, cell cultures were treated with 100 IU/mL IL-2 for various times and luciferase was assayed with the Dual-Glo luciferase system according to the manufacturer’s protocol (Promega Corp.). All transfection experiments were performed in duplicate or triplicate, and the data are presented as the means +/− standard deviations.