Pgp glo assay systems kit

Oxypeucedanin (OPD) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Vincristine sulfate (VCR) was provided by the National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, (NPEC, Nanchang, China). Docetaxel (DTX) was obtained from Xi’an Hao-Xuan Bio-Tech Co., Ltd. (Xi’an, China) Verapamil and rhodamine-123 (Rh-123) were products of Sigma-Aldrich (St. Louis, MO, USA). Trizol RNA extraction kit was purchased from Ambion (Thermo Fisher Scientific, Beijing, China). The reverse transcription kit and P-gp-Glo™ assay systems kit were provided by Promega (Madison, WI, USA). Primary antibodies for mouse monoclonal anti-P-gp (MDR) and mouse monoclonal GAPDH were obtained from Sigma-Aldrich, Inc. and Shanghai Bioleaf Biotech Co., Ltd. (Shanghai, China), respectively.

The ABCB1 ATPase activity was assayed using Pgp-Glo™ assay systems kit (Promega, Madison, WI, USA). The effects in the presence of IXN and positive control verapamil were compared against the basal ABCB1 ATPase activity. IXN (0–30 μM) or verapamil (200 μM) were incubated with recombinant human ABCB1 membranes (25 μg) in the presence of 5 mM MgATP at 37 °C for 40 min. Then, the remaining ATP was detected according to the manufacture’s protocol, using spectraMax M5 (Molecular Devices, Sunnyvale, CA, USA).