Ab78422

A radioimmunoprecipitation assay buffer with a protease inhibitor cocktail (Solarbio, Beijing, China) and 1 mmol/L phenylmethylsulphonyl fluoride was used for the extraction of the total cellular protein from the snap-frozen tissues. A bicinchoninic acid assay (CWBIO, Beijing, China) was applied to the determination of the total protein concentration. Afterward, 10% sodium dodecyl sulfate sulphate polyacrylamide gel electrophoresis was used to separate samples with 100 μg of protein, followed by electrophoretic transfer to nitrocellulose membranes. A blocking buffer (5% skim milk, 0.1% Tween 20®, and 0.01 M tris-buffered saline) was then used to block the membranes at room temperature for 2 hours. Anti-KDM5C (1:100 dilution, ab34718, Abcam, USA), anti-BMP-7 (1:150 dilution, ab56023, Abcam, USA), and anti-BMPRII (1:150 dilution, ab78422, Abcam, USA) were used to incubate the membranes overnight at 4°C. β-actin (1:120,000 dilution, ab6276, Abcam, USA) was used to express all results. Secondary fluorescent antibodies (1:5000 dilution, Odyssey CIX, Lincoln, USA) were then used to incubate the membranes for 2 hours at room temperature, after which an infrared laser scanning imaging system (Odyssey CIX, Lincoln, USA) was used to measure the fluorescence intensity. Based on the ratio of the internal target protein, analysis of protein expression for all target genes was conducted.