To generate the RORγt-GAL4 fusion protein, human RORγt (from 97 to 516) was PCR-amplified from pCDNA2-FLAG-RORγt. The PCR product was ligated into the pFN11A (BIND) Flexi vector (Promega, Madison, WI) containing the yeast GAL4 DNA-binding domain upstream of the cloning site. This vector also expresses the Renilla luciferase under the control of SV40 promotor, allowing normalization for differences in transfection efficiency. The recombination plasmid was named RORγt-GAL4. The vector, pGL4.31 [luc2P/GAL4UAS/Hygro] (Promega, Madison, WI), contains five tandem GAL4 binding sites upstream of a minimal TATA box, which is upstream of a firefly luciferase gene that acts as a reporter for interactions between protein and the ligand.42 (link) The pRL-SV40 Renilla luciferase control reporter vector was purchased from Promega (Madison, WI).
For luciferase activity assay, HEK293T cells were co-transfected with 2 μg ROR-GAL4, 2 μg pGL4.31 and 100 ng pRL-SV40 in a 6 cm dish using Lipofectamine 2000 (Invitrogen, USA). After 24 h of transfection, the cells were incubated with a variety of concentrations of compounds. After 24 h treatment, the cells were lysed with passive lysis buffer and luciferase activities were measured using a Dual-Glo Luciferase Assay System (Promega, Madison, WI) using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA).
For luciferase activity assay, HEK293T cells were co-transfected with 2 μg ROR-GAL4, 2 μg pGL4.31 and 100 ng pRL-SV40 in a 6 cm dish using Lipofectamine 2000 (Invitrogen, USA). After 24 h of transfection, the cells were incubated with a variety of concentrations of compounds. After 24 h treatment, the cells were lysed with passive lysis buffer and luciferase activities were measured using a Dual-Glo Luciferase Assay System (Promega, Madison, WI) using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA).
