Determination of the expression levels of P-gp by western blot analysis

Membrane proteins were harvested by KenGEN Membrane and Plasma Protein Purification Kit (KenGEN BioTECH, Nanjing, China), and the protein concentrations were determined using KeyGEN BCA Protein Quantitation Assay kit (KenGEN BioTECH, Nanjing, China). An equal quantity of protein (48 μg) from membrane protein was resolved using 7.5% SDS-PAGE gel and subsequently transferred onto nitrocellulose membranes (Bio-Trace, Auckland, New Zealand). After blocking the membrane with 5% non-fat milk in Tris-buffered saline (Biotopped) at room temperature for 1 h, the membrane was incubated at 4 °C for 12 h with rabbit monoclonal primary antibodies against MDR1 (1:1250; ab170904) and Anti-Sodium Potassium ATPase antibody-Plasma Membrane Loading Control (1:20,000; ab76020). Antibodies were both purchased from Abcam (Cambridge, United Kingdom). The membranes were incubated with horseradish peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (ZSGB-BIO, Beijing, China) for 1 h and signals were observed using SuperSignal West Pico (Thermo Scientific, Waltham, MA). Western blotting bands intensity was quantified by densitometric analysis using Image J version 2 × (NIH Image Software, Bethesda, MA).