Ab181451

The assay was implemented in line with the previous description [23 (link)]. Generally, the separated proteins were and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, U.S.A.). Then, the membranes were blocked with 5% non-fat milk. Afterwards, the membranes were incubated with primary antibodies, including ZEB1 (1:1000, ab181451; Abcam, Cambridge, MA, U.S.A.), P-gp (1:1000, ab3364; Abcam) and GST-π (1:800, ab47709; Abcam) and GAPDH (1:8000, ab8245, Abcam), followed by probed with corresponding secondary antibody (Abcam) to combine these primary antibodies. At last, proteins blots were assessed with an enhanced chemiluminescence detection kit (Millipore).

Cells were lysed in lysis buffer, and protein concentrations were measured with the BCA protein assay kit (Thermo Fisher Scientific). Proteins were separated by 10% SDS-PAGE and transferred electrophoretically onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated in PBS containing 5% bovine serum albumin for 2 h at room temperature, followed by overnight incubation at 4 °C with different dilutions of the primary antibodies, including antibodies to CDK5 (EP715Y, Abcam, Cambridge, MA, USA), ZEB1 (ab181451, Abcam), E-cadherin (4A2, Cell Signaling Technology, Boston, MA, USA), N-cadherin (D4R1H, Cell Signaling Technology), Twist (ab50581, Abcam), Snail (C15D3, Cell Signaling Technology) and GAPDH (ab9485, Abcam). The membranes were developed with the Odyssey infrared imaging system according to the manufacturer’s instructions. The levels of protein in each sample were normalized relative to those of GAPDH. Each experiment contained triplicate wells of each sample, and all experiments were repeated at least three times.