Gentest atpase kit

The P-gp associated ATPase activity was measured by BD Gentest ATPase kit. The assay was carried out in white opaque 96-well multiplates in triplicate. Recombinant human P-gp membrane (5 mg/mL) was quickly thawed and diluted to 1 mg/mL with assay buffer. Sodium orthovanadate (Na₃VO₄) was used as an ATPase inhibitor. Various concentrations of SSJ26 and SSJ32 were incubated in 20 μg (20 μL) diluted recombinant human P-gp membrane at 37 °C for 5 min. The reaction is initiated by adding 20 mL of 15 mM Mg²⁺ ATP to all wells. At this point, each P-gp reaction contains 5 mM ATP. The plate was incubated at 37 °C for 40 min with brief mixing using a plate shaker. Luminescence initiated by ATP detection buffer. After incubation at 37 °C for 20 min to allow luminescent signal to develop, the untreated white opaque 96-well multiplate was read on luminometer (SpectraMax M5, molecular devices, Sunnyvale, CA, USA). The changes of relative light units (DRLU) were determined by comparing Na₃VO₄-treated samples with SSJ26 and SSJ32 combination-treated groups [26 (link)].

The P-gp or BCRP-associated ATPase activity was measured by BD Gentest ATPase kit. The assay was carried out in white opaque 96-well multiplates in triplicate. Recombinant human P-gp or BCRP membrane (5 mg/ml) was quickly thawed and diluted to 1 mg/ml with assay buffer. Sodium orthovanadate (Na₃VO₄) was used as a ATPase inhibitor. Various concentrations of FLZ or inhibitors(5 µM zosuquidar, 10 µM FTC or 100 µM Verapamil)diluted with assay buffer were incubated in 20 µg (20 µl) diluted recombinant human Pgp or BCRP membrane at 37°C for 5 minutes. Initiate reactions by adding 20 µl of 15 mM Mg²⁺ATP to all wells. At this point, each Pgp reaction contains 5 mM ATP. Mix briefly on a plate shaker or by gently tapping the plate. Incubate for 40 minutes at 37°C. Luminescence initiated by ATP detection buffer. After incubated at 37°C for 20 min to allow luminescent signal to develop, the untreated white opaque 96-well multiplates was read on luminometer (SpectraMax M5, molecular devices, USA). The changes of relative light units (ΔRLU) were determined by comparing Na₃VO₄-treated samples with FLZ and inhibitors combination-treated groups.

[³H]-paclitaxel was purchased from Moravek Biochemicals, Inc. (Brea, CA, USA). Monoclonal antibody C-219 (against P-gp) and loading control antibody BA3R (against β-actin) were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Sipholane analogs were previously synthesized and the structures are shown in Figure 1A and Supplementary Information Figure S1. Fumitremorgin C (FTC) was synthesized by Thomas McCloud Developmental Therapeutics Program, Natural Products Extraction Laboratory, NCI, NIH (Bethesda, MD, USA) and it was a gift from Susan E Bates and Robert W. Robey. The Gentest ATPase kit was purchased from BD Biosciences (San Jose, CA, USA). Doxorubicin, paclitaxel, vincristine, verapamil, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). PAK-104P was a gift of Shin-Ichi Akiyama (Kagoshima University, Kagoshima, Japan) from Nissan Chemical Ind. Co., Ltd. (Chiba, Japan).