Minute plasma membrane isolation kit

MLO-Y4 cells were seeded in 6-well plates with media containing different concentrations of glucose (0, 2.5, 5, 10, 20, and 40 mM) for 24 h. The cells were washed with cold phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) with1 mM polymethylsulfonyl fluoride (ST506, Beyotime) for 10 min on ice. Cell scrapers were used to remove cells from the plates. The cells were collected in lysis buffer, then centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was diluted with cold loading buffer. Protein concentration was determined using the bicinchoninic acid assay kit (P0010, Beyotime). The protein samples were boiled at 100°C for 10 min. Subcellular protein fractions (plasma membrane, organelles, and cytosol) were separated using the Minute plasma membrane isolation kit (SM005, Invent Biotechnologies, Plymouth, MN, USA) according to the manufacturer’s instructions. Plasma membrane protein concentration was analyzed using the bicinchoninic acid assay kit (P0010, Beyotime).

The plasma membrane protein was isolated using the Minute Plasma Membrane Isolation kit according to the manufacture's instructions (Invent Biotechnologies, USA). In brief, the cells were collected and resuspended with lysis buffer A supplemented with Complete™ EDTA-free Protease Inhibitor Cocktail (Roche, #4693116001, Switzerland). The total membrane proteins, including organelles and plasma membrane, were isolated by centrifugation and further analyzed by Western blotting with the indicated antibodies.