Luminescent β-galactosidase Detection Kit II

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Warm enough Reaction Buffer and Reaction Substrate for the entire experiment to room temperature	Add 200 µl of the Reaction Buffer Mixture to each cell lysate and mix gently. Incubate at room temperature (20–25°C) for 60 min

Upstream tips
Warm enough Reaction Buffer and Reaction Substrate for the entire experiment to room temperature
Protocol tips
Add 200 µl of the Reaction Buffer Mixture to each cell lysate and mix gently. Incubate at room temperature (20–25°C) for 60 min

Publication protocol

For mammalian 2-hybrid analysis, CHO cells were cotransfected with a pGL4.27-(UAS)5 reporter plasmid, containing 5 copies of UAS in the LUC reporter vector pGL4.27 (Promega), pCMV-β-Gal, pM-EBIP96(LXXLL) peptide, and VP16-RORα(LBD), or VP16-RORγ(LBD) (2, 49). To measure the activation of the Baml1 and the Gpase6 promoter, Huh-7 cells were cotransfected with pCMV-β-Gal, pCMV10-3xFlag-RORα, and a pGL4.10 reporter plasmid (Promega) containing human Bmal1 (−650/+105) or G6pase (−500/+58) promoter (3), using Lipofectamine 2000 (Invitrogen). For the Il17 promoter analysis, Jurkat cells were cotransfected with pCMV-β-Gal, pCMV10-3xFlag-RORγ or pCMV10-3xFlag-RORα, and a pGL4.14 reporter plasmid under the control of the Il17 promoter (56) and then treated with 20(OH)D3 or 20,23(OH)2D3. After 24 h, the LUC and β-galactosidase activities were measured using a Luciferase Assay Substrate Kit (Promega) and Luminescent β-galactosidase Detection Kit II (Clontech). All transfections were performed in triplicate and repeated at least twice

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