4.6. Western Blot Analysis

The expression of the ABCG2, PI3K - p110β and AKT proteins was determined using Western blotting as previously described [70]^. Briefly, the cell lysates were prepared from the parental S1 and drug-resistant S1-M1-80 cells after incubating with 5 µM of VKNG-2 for 24, 48 and 72 h. After protein quantitation using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), the protein samples were separated by PAGE and then transferred onto PVDF membranes. After blocking the membranes with 5% milk, the membranes were incubated with primary antibodies against ABCG2/PI3k p110β/AKT or β actin (1:1000) at 4 °C overnight, which was followed by further incubation with HRP-linked secondary antibody (1:1000) for 2 h at room temperature. The protein bands were visualized after exposed the membranes to Pierce™ ECL Western blotting substrate (Thermo Scientific, Rockford, IL, USA). The expression levels of the proteins were analyzed by ImageJ software.