Lb940 multilabel reader

The SK1-luciferase constructs were created by inserting an ~0.5 kb and an ~0.6 kb fragment encompassing the predicted binding site into the pGL4-BASIC-luciferase plasmid (Promega, Tokyo, Japan). The primers used to amplify the DNA fragments were SP1_P1 (forward 5′-GGAACCAGCTCGTGGCCCGG-3′ and reverse 5′-TGCTGGGCACGAAGTTCTGG-3′) and SP1_P2 (forward 5′-AGGCTCAGTGCCCTCCCCGC-3′ and reverse 5′-TGCTGGGCACGAAGTTCTGG-3′). A commercial plasmid containing a CMV-driven Renilla reporter system was used as an internal control (Promega). HK-2 cells were plated in six-well plates at 50–70% confluence and were co-transfected with the pCMV-SP1 construct or with an equimolar amount of the empty pCMV vector and the pGL4-SP1-P1 or pGL4-SP1-P2 construct utilizing Lipofectamine 3000 reagents (ThermoFisher). The media was changed 2 h prior to transfection. After the media was changed, the cells were incubated in 10% DMEM for 24 h. The luciferase assays were performed using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega). Briefly, 100 ml of luciferase substrate was added to 20 ml of lysate, and luciferase activity was measured using an LB940 Multilabel Reader (Berthold Technologies, Bad Wildbad, Germany). Each luciferase assay was performed in triplicate.

For BRET measurements HEK 293T cells were trypsinized and plated on poly-lysine-pretreated (0.001%, 1 hour) white 96-well plates at a density of 10⁵ cells/well together with the indicated DNA constructs (0.24–0.3 μg total DNA/well) and the cell transfection reagent (0.5 μl/well Lipofectamine 2000 or 1.5 μl/well GeneCellin). After 6 hours 100 μl/well DMEM containing serum and antibiotics was added. Measurements were performed 24–27 h after transfection. Before measurements the medium of cells was changed to a medium (50 μl) containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl₂, 0.7 mM MgSO₄, 10 mM glucose, and 10 mM Na-HEPES, pH 7.4. Measurements were performed at 37°C using a Mithras LB 940 multilabel reader (Berthold, Germany). The measurements started with the addition of the cell permeable luciferase substrate, coelenterazine h (40 μl, final concentration of 5 μM), and counts were recorded using 485 and 530 nm emission filters. Detection time was 500 ms for each wavelength. The indicated reagents were also dissolved in modified Krebs–Ringer buffer and were added manually in 10 μl. For this, plates were unloaded, which resulted in an interruption in the recordings. All measurements were done in triplicates. BRET ratios were calculated by dividing the 530 nm and 485 nm intensities, and normalized to the baseline.