Atpase assay kit

ATPase activity from MP, MPγ, EV and EVγ was measured using an ATPase assay kit according to the manufacturer’s instructions (Sigma-Aldrich). A phosphate standard was used for creating a standard curve. MP, MPγ, EV and EVγ (1 × 10¹¹ particles/mL) were incubated with 4 mM ATP for 60 min at room temperature in assay buffer with malachite green reagent. The formation of the colorimetric product that forms in the presence of free phosphates was measured with a spectrophotometer at 620 nm. As a control for possible phosphate contamination, samples were incubated in assay buffer without ATP. The signal from these samples was subtracted from the measurements.

The ATPase activity was measured using the ATPase assay kit (Sigma-Aldrich), according to the protocol of Sigma-Aldrich. In brief, 10 μL of the concentrated samples were dispensed into microplate. Ten microliter of 4 mM ATP was added in 20 μL assay buffer and incubated at room temperature for 30 min. Two hundred microliter of the reagent was added to each well and was incubated for 30 min at room temperature. Readings at 600–650 nm was taken with enzyme-linked immunosorbent assay plate reader (Vmax).

Tetrandrine with over 98% purity was purchased from Shanghai Yuanye Biotechnology Company (Shanghai, China), while monoclonal anti-P-glycoprotein antibody, doxorubicin, paclitaxel, vincristine, cisplatin, verapamil, MTT [3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide], ATPase assay kit, ABCB1 antibody (catalogue number p7965) and other chemicals were purchased from Sigma (St. Louis, MO, USA). Alexa flour 488-conjugated goat anti-mouse IgG was purchased from Molecular Probes (Eugene, OR, USA) and [³H]-paclitaxel (15 Ci/mmol, MT552) was purchased from Moravek Biochemicals (Brea, CA, USA). Mammalian Protease Inhibitor Cocktail 100X (AMRESCO, LLC), HRP-labeled rabbit anti-mouse secondary IgG antibody was purchased from Santa Cruz (Dallas, TX, USA), and BD Pharmingen^TM PI/RNase Staining Buffer were purchased from BD Biosciences (Franklin lake, NJ, USA). Monoclonal anti-GAPDH antibody, Reverse Transcriptase reagent for RT-PCR SuperScript^® II, TRIzol^® Reagent, Pierce^TM BCA protein Assay Reagent A/B, and Pierce^TM ECL Western blotting substrate were purchased from Thermo Fisher (Rockford, IL, USA), while sodium orthovanadate was purchased from Alfa Aesar (Ward Hill, MA, USA). Adenosine triphosphate (ATP) was purchased from Amresco (Solon, OH, USA).