Mg8600

Total proteins were extracted from HepG2 cells and Huh7 cells using radio-immunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China). After protein quantitation using the bicinchoninic acid assay, the proteins were separated by PAGE with a 5% stacking gel and a 10% separating gel and then transferred onto a nitrocellulose membrane using wet transfer apparatus. Next, the membrane was blocked with 5% BSA for 1 h and incubated with the diluted rabbit anti-human antibodies from Abcam (Cambridge, UK) at 4°C overnight: MDR-1 (ab129450, 1:1,000), MRP2 (ab203397, 1:500), MRP3 (ab107083, 1:250), GAPDH (ab37168, 1 μg/mL), and E-cadherin (ab15148, 1:500). Next, the membrane was washed using PBS/Tween-20 and further incubated by applying 5% skimmed milk-diluted secondary antibody goat anti-rabbit IgG (ab205718, 1: 5000) for 1 h in avoidance of light. Subsequently, the protein bands were developed and visualized in a gel imaging system (MG8600, Bio-Rad, Hercules, CA, USA). Images were analyzed using the Image-Pro Plus software (Version 7.0, Media Cybernetics, Silver Springs, MD, USA). The expression levels of target proteins relative to GAPDH were calculated.

The cells (NCI-H1975 or A549 at 80% confluence in 6-well plates) were lysed with RIPA buffer (Thermo Fisher Scientific, Inc.) and the protein concentration was determined using the bicinchoninic acid kit (Beyotime Institute of Biotechnology). Proteins (20 µg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (MilliporeSigma). Following blocking (with 5% nonfat milk in TBST at room temperature for 1 h), the membrane was incubated overnight at 4°C with the primary antibodies: PTEN (1:1,000; cat. no. ab267787), p-Akt (1:500; cat. no. ab131443), Akt (1:10,000; cat. no. ab179463), p-PI3K (1:500; cat. no. ab182651), PI3K (1:1,000; cat. no. ab191606) and GAPDH (1:1,000; cat. no. ab9485; all Abcam). Subsequently, PBST was used to rinse the membranes three times. Secondary antibody goat anti-rabbit IgG H&L (HRP; cat. no. ab6721; Abcam) was used to incubate the membranes at room temperature for 1 h. All protein bands were visualized using ECL chemiluminescence (Thermo Fisher Scientific, Inc.). Protein bands were visualized in a gel imaging system (MG8600, Bio-Rad). Images were using Image Pro Plus software (Version 7.0, Media Cybernetics).