Bright glotm luciferase assay kit

Luciferase assay for PPARγ activity was conducted with PPARγ luciferase construct (a kind gift of Dr. B. Spiegelman, Addgene #1015) using BrightGlow (Promega, WI, USA) and measured with a GLOMAX 20/20 Luminometer. Transfections were carried out on MC3T3 E1 cells (control) or cells with stable Rb1 knockdown using Superfect (Qiagen) according to manufacturer recommendations. Forty-eight hours after transfection, cells were trypsinized, pelleted, and then resuspended in serum free media. An aliquot was used to count cells for normalization, the rest was assayed using Promega’s Bright-GloTM Luciferase Assay Kit (Promega, cat. no. E2610). Equal volumes of the resuspended cells and Bright-GloTM were mixed and incubated for 2 min. Samples were read for 10 s, each after a 2 s delay. Luciferase readings for each of the samples were then normalized according to the number of cells per milliliters. All measurements were carried out on triplicate samples, and experiments were repeated at least thrice.

TCDCA (purity ≥ 98%) and TLCA (purity ≥ 97%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). INT-777 (purity ≥ 98%) was purchased from MedchemExpress Co. (Princeton, NJ, USA). Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was purchased from Beyotime Co. (Beijing, China). pMD19-T vector, Hind restriction endonuclease III (Hind III), restriction endonuclease XbaI, and SYBR^® Premix Ex Taq^TM were purchased from Takara Co. (Shiga, Japan). pCMV-C-EGFP plasmid and pCMV-Blank plasmid were purchased from Tiangen Co. (Beijing, China). Lipofectamine^TM 2000 transfection reagent was purchased from Invitrogen Co. (Carlsbad, CA, USA). Sheep anti-rabbit TGR5 antibody, Rabbit anti-goat TGR5 antibody, and Rhodamine-labeled mouse anti-goat antibody were purchased from Cruz Santa Co. (Dallas, TX, USA). Horseradish peroxidase-labeled Goat anti-rabbit antibody was purchased from KPL Co. (Gaithersburg, MD, USA). cAMP ELISA Kit was purchased from Cayman Co. (Ann Arbor, MI, USA). The Bright-Glo^TM Luciferase Assay Kit was purchased from Promega Co. (Madison, WI, USA). The Nuclear Extract Kit was purchased from Active Motif Co. (Carlsbad, CA, USA). Dulbecco’s Modified Eagle Medium was purchased from Hyclone Co. (Shanghai, China). Fetal bovine serum was purchased from ExCell Co. (Taicang, China). Other reagents were purchased from ExCell Co.