Hybond p polyvinylidene fluoride membranes

OP9 cells were pretreated with 40 μg/mL AOE for 1 h and then differentiated at 37°C. Cells were lysed with ice-cold mammalian protein extraction reagent (M-PER) (Pierce Biotechnology, Rockford, IL, USA), and the protein concentration in the lysate was determined using the Bradford method. Samples (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 10% acrylamide and transferred to Hybond-P polyvinylidene fluoride membranes (GE Healthcare Life Sciences, Buckinghamshire, UK) using a western blot apparatus. Each membrane was blocked for 2 h with 2% bovine serum albumin or 5% skim milk and then incubated overnight at 4°C with 1 μg/mL of a 1 : 2,000 dilution of primary antibody. HRP-conjugated IgG (1 : 2,000 dilution) was used as the secondary antibody. Protein expression levels were determined by signal analysis using an image analyzer (Fuji-Film, Tokyo, Japan).

Cell pellets were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate and 1% Triton X-100) supplemented with protease inhibitor cocktail (Complete, Roche Applied Science, Penzberg, Germany). Protein concentrations were determined with Coomassie brilliant blue (Thermo Scientific, Darmstadt, Germany). Protein samples (10–25 µg of total protein per lane) were separated by SDS-PAGE and transferred to Hybond-P polyvinylidene fluoride membranes (GE Healthcare, Boston, MA, USA). After blocking in 5% nonfat dry milk in TBS-T (0.1% Tween 20) for 1 h immunoblots were probed with primary antibodies (Table S5) over night at 4 °C followed by an incubation of peroxidase-conjugated secondary antibody (GE Healthcare, Boston, MA, USA) for 1 h according to the manufacturer’s protocol Blots were detected using WesternBright Chemiluminescence Substrate (Biozym, Hessisch Oldendorf, Germany) and the digital imaging system from Intas (Göttingen, Germany). All Western blot images have been cropped for improved clarity and conciseness. Quantification by densitometry was performed using Image J software (National Institutes of Health, Bethesda, Rockville, MD, USA). Relative band intensities were expressed as arbitrary units and normalized to the corresponding actin.