Anti abcb1

The transfected cells were collected, total proteins were extracted, and the protein concentration was quantified using the BCA Protein As-say Kit. Membranes were incubated with primary antibody (anti-ABCB1, anti-ABCC1 and anti-ABCG2 (Novus Biologicals, USA), anti-PTEN (Abeam, USA), anti-p-Akt, anti-t-Akt, anti-p-mTOR and anti-t-mTOR (Cell Signaling Technology, USA) and anti-β-actin (Proteintech, USA) at 4 °C overnight. Then membranes were incubated with anti-rabbit/mouse IgG secondary antibody (Jackson, USA). Western blot analysis were performed as described in previous study [23 ].

Cells were washed twice with ice-cold PBS and lysed with cell lysis buffer containing 5 μL/mL protease inhibitor cocktail in IGEPAL. Lysates were syringed with 23-gauge needles to shear cellular DNA and centrifuged at 12,000× g rpm at 4 °C for 5 min. Total cell protein concentrations of the resulting supernatants were determined using BCA protein assays. Equal amounts of cell protein were loaded onto 10% (v/v) acrylamide gels for separation by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes, blocked for 1 h using 5% (w/v) skim milk or 0.1% (w/v) BSA in PBS-Tween (0.05% v/v), then incubated with anti-ABCB1 (1:2000 (Novus Biologicals), overnight 4 °C) or anti-tubulin (1:3000, overnight 4 °C) antibodies. Membranes were rinsed with PBS-Tween, then incubated with HRP-conjugated anti-mouse secondary antibodies (1:10,000) for 1 h at room temperature, and then rinsed again with PBS-Tween. Protein bands were visualised via chemiluminescence and the Bio-Rad ChemiDoc imaging system.