Anti ha antibody

The membrane-impermeable biotinylation reagent NHS-SS-biotin was utilized to examine the cell surface level of OATP1B1 and mutants. Briefly, HEK293 cells expressing OATP1B1 or mutants were labeled with 0.5 mg/mL of NHS-SS-biotin. After dissolving the cells with RIPA buffer, streptavidin-agarose beads were added to bind the biotin-labeled membrane proteins. The bound proteins were then released in Laemmli buffer, loaded onto the SDS-polyacrylamide electrophoresis gel, transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA), and finally, detected with anti-HA antibody (Beyotime Biotechnology, Jiangsu, China). Actin and integrin were used as loading controls for total and membrane proteins, respectively. Three independent experiments were performed for each analysis, and one representative blot is shown.

The collected cell samples were lysed in RIPA lysis buffer (ComWin Biotech Co, Beijing, China) prepared with protease inhibitors (Roche, Basel, Switzerland), and then were placed on ice for 30 min. The lysed cells were centrifuged at 12,000×g for 20 min at 4 °C, and the supernatant were collected. Protein concentrations were determined by the BCA Protein Assay Kit (Thermo Fisher Scientific, CA, USA). The supernatant (20 μg/well) was mixed with 5 × SDS-PAGE loading buffer and boiled in a 100 °C water bath for 5–10 min. The protein samples were subjected to gel electrophoresis and then transferred to PVDF membranes. After blocking with 10% skimmed milk powder for 2 h at room temperature, the membranes were incubated with anti-parkin antibody (1:1000, Millipore, Massachusetts, USA), anti-DMT1 antibody (1:800, OriGene, Rockville, MD, USA), anti-β-actin antibody (1:10,000, Bioss, Woburn, MA, USA), anti-HA antibody (1:2000, Beyotime Biotechnology, Shanghai, China), anti-Myc antibody (1:2000, Beyotime Biotechnology, Shanghai, China) and anti-Flag antibody (1:2000, Beyotime Biotechnology, Shanghai, China) overnight at 4 °C. Membranes were incubated with a corresponding secondary antibody (1:10,000, Bioss, Beijing, China) for 1 h at room temperature. Finally, blots were imaged using a BioSpectrum Imaging System (UVP, Upland, CA, USA) and quantified using ImageJ software.

Co-immunoprecipitation was performed following standard procedures. In brief, the cells were washed in PBS and lyzed in cold NP-40 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40) supplemented with a protease/phosphatase inhibitor cocktail (Cell Signaling Technology). The lysate was briefly sonicated and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was then collected and 1 mg of total protein lysate was incubated with protein A-conjugated agarose beads (Beyotime technology, P2051) and primary antibodies overnight at 4°C. Anti-FLAG antibody (Beyotime biotechnology, AF519, 1: 250 dilution) and anti-HA antibody (Beyotime biotechnology, AF0039, 1:250 dilution) were used. The beads were spun down, washed with PBS buffer, and denatured with 2× Laemmli sample buffer (Bio-Rad), followed by Western blotting for validation.