Iba7300

Bm12 cells at logarithmic growth phase were used for transfection. Plasmid DNAs were mixed with Lipfectamine 2000 (Invitrogen) and added to cells in each well of 12-well culture plates with Grace medium (Invitrogen). To normalize the firefly luciferase activity, the renilla luciferase vector, pRL-SV40, was co-transfected with each of the pGL3-drived vectors containing tested promoters. After 6-h post transfection, the old medium was replaced with fresh Grace medium containing 10% FBS. The cells were cultured for additional 48 h at 28 °C before promoter activity assay. The cells were washed once with filtered PBS and then lysed in 200 μL Passive Lysis Buffer (Promega). Luciferase activity of the supernatant was analyzed using the Dual-Luciferase Assay System (Promega) according to the manufacturer’s instruction with a luminometer (IBA7300, Veritas, Turner Biosystems). All assays were conducted three times.

Bm12 cells at their logarithmic growth phase were inoculated in culture media in 12- or 24-well culture plates (Corning, New York, NY, USA) and cultured for 12 h. Cell transfection and co-transfection were conducted when the cells were at approximately 80% density. Plasmid DNAs were mixed with Fugene HD transfection reagent (Promega, Madison, USA) and added to cells in each well of 12- or 24-well culture plates with Grace medium (Invitrogen). To normalize the firefly luciferase activity, the renilla luciferase vector, pRL-SV40, was co-transfected with each of the pGL3-derived vectors containing tested promoters. The cells were cultured for additional 48 h at 28 °C, followed by the luciferase activity assay, protein or RNA isolation.
For luciferase activity measurement, the cells were washed twice with filtered PBS and then lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI, USA). The samples were centrifuged at 800g for 5 min at room temperature. The supernatant was used to analyze the luciferase activity using the Dual- Luciferase Assay System according to the manufacturer’s protocol with a luminometer (IBA7300, Veritas, Turner Biosystems). The luciferase activity was normalized to the renilla luciferase activity. All assays included three biological replications and three technical replicates. The luciferase activity was represented as mean ± standard error (SE).