Centro lb960 96 well luminometer

HEK293 cells were seeded in 24-well plates at a density of 1 × 10⁵. After 24 h, the cells were transfected using Lipofectamine 2000 (Invitrogen). The promoter region of FGF4 targeted by Sox2 was cloned into the pGL3-Luc vector (Promega, Madison, WI, USA), and subsequently co-transfected with Myc-Sox2 and Flag-OTUD7B or Flag-COP1, DET1 and CUL4A, together with the pRL-SV40 Renilla luciferase construct. Single overexpression of Sox2 was used as controls. Cell extracts were prepared 48 h after transfection and the luciferase activity was measured by using the Dual Luciferase System (Promega, Madison, WI, USA) and a Centro LB960 96-well luminometer (Berthold Technologies, Bad Wildbad, Germany).
The promoter region of FGF4 targeted by Sox2 is as below:
5′AGACCGTCTTTTAGAAAATAACAAGAAGAAAAGACATTTCAACTGTCTTCTCCCCAACACTCTTGGAGCCTAGGGCCTGGATTTAAAAAACACAAAATCTTATTGTCCTGTGAGCCACCAGACAGAAAGGAAGTTTGGGAGGAGCTCCCACCTCAGCTTTGGGGTGTGGCTTCTTCCATCTTGGGCTGTGGTACAGAATAGTATTTTAAGTATCCCATTAGCATCCAAACAAAGAGTTTTCTAAAGGAATGTGAAAGACAAAAAAAAAAAAATGCCAATGAGATTTTCCAGTCTTGCTGTCTGTAGCCTCCCATAAAGTTAATTCGGGAGGTTGCTCAGAAGTCTCCCAGCAGAGTCTTA-3′

CHO cells were cultured in Dulbecco's modified Eagle's medium with high glucose (Invitrogen), 10% standard fetal bovine serum (Hyclone), and 1% penicillin/streptomycin and maintained at 37°C with 5% CO₂. For a 24-well plate, 0.5 µg of EO plasmid, 0.5 µg of MS plasmid, and 0.3 µg of pGL3 NSO plasmid were co-transfected per well using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Renilla luciferase plasmid (0.05 µg; pRL-TK from Promega) was co-transfected as an internal control. Firefly and Renilla luciferase activities were measured 24 h post transfection using the Dual Luciferase Kit (Promega) and a Centro LB960 96-well luminometer (Berthold Technologies). Alternatively, F9 embryonal carcinoma (EC) cells were used in the luciferase assay for motif characterization to understand the importance of variable positions in the Sox/Oct motif sequence. Cell culture, transfection, and sample preparation for F9 EC cells were performed as described earlier [31 (link)].

The promoter regions including the Zfp206-binding sites for IL2, IL12b and IL23a were cloned into pGL3-basic vector (Promega). A dual luciferase system (Promega, Madison, WI, USA) was used. For the luciferase assay in 293T cells, 4 × 105 cells were seeded into one well of a 24-well plate. After 18 h, 275 ng of luciferase reporter plasmid, 1 ng of plasmid pRL-SV40 and either 1 μg of NFATc2 overexpression vector or empty vector were co-transfected into the cells using Lipofectamine 2000 (Invitrogen). The pRL-SV40 plasmid served as an internal control for normalizing the transfection efficiency. After 40 h of transfection, the 293T cells were lysed, and the luciferase activity was determined with the dual luciferase system (Promega) using a Centro LB960 96-well luminometer (Berthold Technologies).