Anti p gp

Cells were lysed with sodium dodecyl sulfate (SDS) lysate containing 1% benzyl sulfonyl fluoride and 1% phosphatase inhibitor. After carrying out ultrasonic cracking, the total proteins were run in SDS-polyacrylamide gel electrophoresis. The primary antibodies of the rabbit anti-P-gp (1:50,000 dilution, Cell Signaling Technology, Inc., Boston, MA, USA), pro-apoptotic protein Bax (1:1,000 dilution, Proteintech, Wuhan, China), anti-apoptotic protein Bcl-2 (1:1,000 dilution, Cell Signaling Technology), and mouse anti-β-actin (1:50,000 dilution, Proteintech) and the secondary antibodies of goat anti-rabbit IgG and anti-mouse IgG labeled with horseradish peroxidase (both 1:10,000 dilution, Proteintech) were used. The protein bands were photographed by the chemiluminescence imaging system (Tanon Science & Technology, Shanghai, China).

Cells were treated with chemicals under study followed by protein extraction using lysis buffer (50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) which is quantified by Pierce BCA Protein Assay Kit (Thermo Fischer Scientific, USA). Deglycosylation was performed prior to electrophoresis using PNGase F (New England Biolabs) for heavily glycosylated proteins. Equal amounts of protein extracts were electrophoresed on 8% and 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel followed by transfer to nitrocellulose membrane. The membranes were incubated overnight at 4 °C with anti-COX-2 (Abcam, Cambridge, MA), anti-P-gp (Cell signaling technology, MA, USA), anti-EP1 (Abcam, Cambridge, MA), anti-Vinculin (Santa Cruz Biotechnology, CA, USA) and anti-HSC70 (Santa Cruz Biotechnology, CA, USA) antibodies. After incubation with horseradish peroxidase-conjugated secondary antibodies (GeNei, Bangalore, India; Santa Cruz Biotechnology, CA, USA), the bands were visualized using ECL detection reagents (Pierce) on Chemi Doc™ MP Imaging system (Bio-Rad, Hemel Hempstead, UK) and quantified with the AlphaImager 3400 (Alpha InnoTech corporation, Sam Leandro, CA) software. The intensities of the test genes under study were normalized using either vinculin or HSC70.

Total proteins were extracted from cultured cells using SDS lysis buffer (Beyotime, Shanghai, China). Equal amounts of protein were analyzed by 8% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% non-fat milk at room temperature for 1 h, membranes were incubated with the following primary antibodies overnight at 4°C: anti-P-gp (1:1000; Cell Signaling Technology, United States), anti-MRP1 (1:500; Abcam, United States), anti-BCRP (1:500; Millipore, United States), anti-α-tubulin, and anti-β-actin (1:5000; Proteintech Group, United States). The appropriate peroxidase-conjugated antibodies (anti-mouse or anti-rabbit) were then incubated with the membranes at room temperature for 2 h. Reactive bands were detected using ECL-Plus reagent (Merck Millipore, Darmstadt, Germany), and the band intensity was quantified using a Bio-Rad 2000 gel imaging system equipped with QUANTITY ONE software (Bio-Rad Laboratories, Hercules, CA, United States).

Both type of cells, CMT-7364/S and CMT-7364/R were plated in a 6-well plate at 1 × 10⁶ cells per well. After fully attachment, protein extraction was performed with ice-cold lysis buffer (P0013B, Beyotime, China) following the manufacturer instructions, and then proteins were quantified using the BCA protein assay kit (P0012S, Beyotime, China). Equal protein amount (20 μg protein per lane) were seperated on a 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH000 10, Merck Millipore). The membranes were blocked with 5% skim milk diluted with TBST for 1 h at room temperature, followed by incubation with primary antibody overnight. Primary bodies included anti-P-gp (1:1,000; Cell Signaling Technology, 13342), anti-GAPDH (1:2,000; Cell Signaling Technology,5174), anti-E-Cadherin (1:1,000; Cell Signaling Technology, 3195S), anti-Vimentin (1:1,000; Cell Signaling Technology, 5741S), anti-MUC1-N (1:2,000; Proteintech, 19976-1-AP), anti-BCRP (1:2,000; Proteintech, 27286-1-AP). Subsequently, membranes were incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-rabbit IgG (1:5,000; Cell Signaling, 7074) for 1 h at 37°C Finally, using a chemiluminescence imaging analysis system (Tanon 5200, China) to detect signals. The quantification of the bands was done by Image J software.