Passive lysis buffer of the dual luciferase reporter assay kit

HEK293 cells were seeded in 24 well plate in 1 ml/well of DMEM 10% FCS. 24 h later, medium was changed to a fresh 0.5 ml/well, and cells were transfected using jetPEI reagent (101-01N, Poly-plus transfection Inc.), according to manufacturer's instructions. Briefly, 200 ng/well of the expression plasmid (pCMV-empty/E2F1), 200 ng/well of the firefly luciferase reporter plasmids (ASK1^WT/ASK1^m1/ASK1^m2/ASK1^m3), and 2 ng/well of the Renilla luciferase reporter plasmid were used for each transfection. 4 h after transfection, medium was changed to fresh DMEM 10%FBS with or without TNFα (10 ng/ml) and with or without SP600125. Cells were lysed 24 h after incubation with or without TNFα 10 ng/ml and with or without SP600125 treatments by applying 100 μl of Passive Lysis Buffer of the Dual Luciferase Reporter Assay Kit (E-1910, Promega Corporation) into each well of the 24-well plate. 20 μl of cell lysate were used for the luciferase reporter assay with the same kit, according to the manufacturer's protocol. Luminescence intensity was quantified in a GloMax 20/20n Luminometer (Promega Corporation). The experiments were performed at least in triplicate. As a control for transfection efficiency, the firefly luciferase activity values were normalized to the Renilla luciferase activity values.

HEK293 cells were seeded in 24-well plates in 1 ml/well of DMEM 10% FCS. 24 h later, medium was changed to fresh 0.5 ml/well, and cells were transfected using jetPEI reagent (Poly-plus transfection, 101-01N), according to the manufacturer's instructions. Briefly, 200 ng/well of the expression plasmid (pCMV-empty/E2F1), 200 ng/well of the firefly luciferase reporter plasmids (Promega Corporation, E-1761) containing BECN1, ATG7, ATG12, DRAM1 or MAP1LC3B, and 2 ng/well of the Renilla luciferase reporter plasmid were used for each transfection. Four h after transfection, the medium was changed to fresh DMEM 10% with or without TNF (10 ng/ml). Cells were lysed 24 h after incubation with or without TNF 10 ng/ml treatment by applying 100 µl of Passive Lysis Buffer of the Dual Luciferase Reporter Assay Kit (Promega Corporation, E-1910) into each well of the 24-well plate. 20 µl of cell lysate were used for the luciferase reporter assay with the same kit, according to the manufacturer's protocol. Luminescence intensity was quantified in a GloMax 20/20n Luminometer (Promega Corporation, Madison, WI). The experiments were performed at least in triplicate. As a control for transfection efficiency, the firefly luciferase activity values were normalized to the Renilla luciferase activity values.