Centro xs3 lb960 high sensitivity microplate luminometer

A split luciferase assay was conducted in accordance with the protocol provided by Promega Corporation (WI, USA). HEK293T cells were transfected with V5_LAR_FRBLgBiT/Myc_LAR_FKBPSmBiT-expressing plasmids or control V5_LAR_LgBiT/Myc_LAR_SmBiT-expressing plasmids so that the total DNA amount was 100 ng/well. After transfection, the cells were cultured at 37°C under 5% CO₂ for 20∼24 h. Then, before application of the substrates, the culture media were exchanged with Opti-MEM^® I (1X) (Gibco^® by Life Technologies^TM, Thermo Fisher Scientific) and incubated at ambient temperature for 10 min. The transfected wells were recorded for at least 5 min before substrate addition to assess the basal status. Luciferase substrate (Promega Corporation) was applied for 5 min before the generated luminescence was recorded for 40 counts. Then, vehicle or rapamycin (20 nM) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was applied, and the subsequent changes in luminescence were recorded for 61 counts. The interval between counts was 1 min. The whole process was performed at ambient temperature. Luminescence was detected using a Centro XS3 LB960 High Sensitivity Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany).

3′-UTR fragments of interest were cloned downstream of the Renilla luciferase gene into the psiCHECK-2 vector (Promega, Fitchburg, WI, USA). The empty Firefly luciferase reporter was used as control. Cortical neurons (100,000 cells per 24-well) were transfected with 8 µg of reporter plasmid (6 wells of a 24-well plate) using calcium transfection. Luciferase activity was measured 24 h after transfection using a Centro XS3 LB 960 High Sensitivity Microplate Luminometer (Berthold, Bad Wildbad, Germany). Ratios of Renilla/Firefly luciferase activity were calculated and normalized to the luciferase empty vector values. A minimum of 3–6 independent experiments were performed. The normalized values from independent experiments were used to determine significant differences using the student’s t-test.