Luciferase Reporter Assay in Cell Lines

RTG2 and RTgill cells (3 × 10⁴) were plated in a 12-well dish and transfected 72 h post plating with indicated plasmids for 48 h, washed with PBS twice and then lysed for 15 min at 4 °C with 100 µL of 1X passive lysis buffer (Promega, Madison, WI, USA). In total, 50 µL of the clarified lysate was combined with 50 µL of luciferase buffer (250 mM Glycylglycine, 200 mM DTT, 100 mM ATP, 200 mM luciferin) and relative luminescence values determined using Molecular Device SpectroMax ID5 (Molecular Devices, LLC, San Jose, CA, USA). Luciferase values were normalized to total protein content of each sample determined by Bradford assay. Bradford assay was performed by adding 2 µL of lysate to 100 μL of 1X Bio-Rad Protein Assay Dye reagent concentrate (Bio-Rad, Hercules, CA, USA). Samples were briefly mixed and absorbance was read at 595 nm.