Anti p jnk

Western blotting was used to investigate the protein expression of BMP9, ERK, P38 and JNK. The protein samples were extracted from brain tissues or astrocytes using RIPA lysis buffer (BioTeke Corporation, Beijing, China) and then protein levels were determined using the bichioninic acid method. A 20 µg protein sample was subjected to 12% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes. Blots were blocked with 5% non-fat milk solution for 1 h at room temperature and then incubated overnight with anti-BMP9 (cat. no. sc514211; 1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-p-ERK (cat. no. BS74621; 1:2,000; Bioworld Technology, Inc., St. Louis Park, MN, USA), anti-p-P38 (cat. no. BS6381; 1:2,000; Bioworld Technology, Inc.), or anti-p-JNK (cat. no. Bs4763; 1:2,000; Bioworld Technology, Inc.) at 4°C followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (cat. no. ZB2301; 1:3,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) for 1 h. The blots were visualized with the ECL system (GE Healthcare Life Sciences, Little Chalfont, UK) and the intensity of each band quantified by using the Chemi Doc XRS system.