Lightswitch dual assay kit

HEK293 cells were stably transfected with either empty vector (pCMV6) or Sox9 expression vector (Origene). These cells were then utilized for promoter luciferase reporter assays^{70 (link)}. Briefly, 5 × 10³ cells were plated overnight on white poly-l-lysine-coated 96-well plates, followed by transient transfection with either promoter constructs (Switchgear Genomics, encoding 2-kb sequence upstream of transcription start sites of the following genes: Gadd45a, Wwp2, Sema3e, and Myof) or empty promoter construct at 30 ng in combination with the Cypridina TK control construct (Switchgear Genomics) at 1 ng, according to the manufacturer’s protocol (Switchgear Genomics, Lightswitch Dual Assay kit, DA010). The promoter construct encodes a Renilla luminescent reporter gene, called RenSP, while the transfection and normalization vector encodes a Cypridina luciferase. The Renilla luciferase activity was normalized with the Cypridina luciferase activity.