Abcg1

The protein was extracted with RIPA lysate and protease inhibitor 100:1, and the BCA kit was used for protein quantification. After adding the loading buffer, the protein was boiled and stored at − 20 °C. The preserved protein samples were electrophoresed on a 6% SDS-PAGE gel at 80V 120 min; then 250 mA 150 min, electro-transported to the PVDF membrane; the skimmed milk powder was blocked for 1.5 h with rabbit anti-ABCA1 (1:1000, CST, USA), ABCG1 (1:500, Proteintech, Wuhan, China), SR-BI (1:500, Sangon Biotech, Shanghai, China) and GAPDH (1:1000, Sangon Biotech, Shanghai, China). Incubate overnight in a shaker at 4 °C, add horseradish peroxidase (HRP) labeled goat Anti-rabbit IgG (1:1000, Beyotime, Shanghai, China), using chemiluminescence western blot detection system to detect protein expression. Using GAPDH as an internal reference, the development results were analyzed for gray data results.

The cells were washed with precold PBS three times, and total and fractionated cellular proteins were isolated. The cytoplasmic and nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN Biotech). The membrane protein was extracted using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. After protein content determination, extracted proteins were separated by using 10% SDS-PAGE gel and then transferred onto nitrocellulose membranes using a Bio-Rad transfer blotting system. The membranes were incubated with primary antibodies against ABCG1 (Proteintech, USA), Nox4 (Santa Cruz Biotechnology, USA), HO-1 (Santa Cruz Biotechnology, USA), Nrf2 (Cell Signaling Technology), and p47phox (Santa Cruz Biotechnology, USA) antibodies. Proteins were visualized using an enhanced chemiluminescence detection system (ECL, Cell Signaling Technology Inc.). Anti-β-actin, anti-lamin B1, anti-Na+/K+ATPase (Santa Cruz Biotechnology, USA) were used to control for equal protein loading.

Oxaliplatin, and Src inhibitor saracatinib (AZD0530) were used for the construction of drug-resistant cell lines, and other anti-cancer molecular targeting drugs were purchased from ApexBio (Houston, TX, USA) and Selleck (Houston, TX, USA). Monoclonal antibodies to the following proteins were used in western blot: E-cadherin, vimentin, PCNA, FZD8, DKK1, AXIN2, WNT6, and β-catenin (purchased from Abcam, Cambridge, MA, USA) and p-LRP6, GSK-3β, AXIN2, cyclin D1, SRC, OCT4, ABCG1, and BCL-2 (purchased from Proteintech, Chicago, IL, USA).