Luciferase cell culture lysis buffer

NIH3T3 cells were plated at a density of 8 × 10⁴ cells/ml in 24 well plates and cultured as described above. Cells were transfected the next day with Lipofectamine and Plus reagent following the manufacturer’s protocol (Invitrogen). Each transfected well received 0.05 μg of GATA4 expression vector, 0.2 μg of steroidogenic gene promoter–luciferase reporter construct, and 0.025 μg of plasmids expressing NotchICDs or Hey proteins. All transfections included 0.05 μg of pCMV-eGFP (Invitrogen, Carlsbad, CA). Total plasmid DNA was kept at 0.4μg/well in all transfections with the addition of empty vector. All assays were done in triplicate on the same plate. The control sample for these assays was transfection of the reporter only. At 48 h post-transfection, cells were lysed in Luciferase Cell Culture Lysis Buffer (Promega, Madison, USA). Luciferase activity was measured for each well by reacting 20 μL of cell lysate with 100 μL of Luciferase Assay Buffer (Promega) in 96-well plates, using an FLx800 microplate reader (Biotek Instruments, Winooski, USA) all samples were normalized to GFP expression. All data are the mean +/− s.d. of three experiments done in triplicate, and statistical significance was determined using a one-way ANOVA.