Ecl fluorescence detection kit

After the total protein content was extracted using a RIPA lysis buffer (R0010, Solarbio) containing PMSF, an estimation of the protein concentration was determined using a BCA kit (Thermo Scientific, USA). After the sample was mixed with the loading buffer solution, 5 µg of protein was added to each well, followed by electrophoresis at 80 V for 2 hours. The protein was electro-blotted to a polyvinylidene fluoride (PVDF) membrane (ISEQ00010, Millipore, Billerica, MA, USA) at 110 V for 2 hours. After 2 hours of blocking with 5% nonfat milk at 4 °C, the membrane was probed with primary rabbit antibodies to RUNX2 (ab236639, 1:1,000, Abcam, UK), TGFBR2 (ab186838, 1:1,000, Abcam, UK) and GAPDH (ab8245, 1:2,000, Abcam, UK) overnight at 4 °C. The membrane was then cultured with HRP-labeled goat anti-rabbit immunoglobulin G (IgG) antibodies (Beijing Zhongshan Biotechnology Co., Ltd., 1:5,000) at room temperature for 1 hour. After the fluorescence imaging of membrane by an ECL fluorescence detection kit (number BB-3501, Amersham, UK) in gel imaging system, a Bio-Rad image analysis system (Bio-Rad Company, USA) was adopted to take photos of the membrane, and ImageJ software was employed to analyze the obtained results. The relative protein content was shown by the gray values of corresponding protein bands/GAPDH protein bands.