Protease and phosphatase inhibitor cocktail

Proteins were extracted from cells with RIPA buffer with a protease and phosphatase inhibitor cocktail (Keygen, Nanjing, China). After the determination of concentrations using BCA, the proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes. The blots were treated with anti-ITGB1 (1:1000) (ab30394, Abcam), anti-MMP2 (1:2000) (ab92536, Abcam), anti-AKT (1:1000) (4685, CST), anti-P-AKT (1:1000) (4228, CST), anti-PI3K (1:1000) (4249, CST), anti-P-PI3K (1:1000) (4228, CST), and anti-GAPDH (1:5000) (ab181602, Abcam). The bands were visualized using enhanced chemiluminescence. The loading control was GAPDH.

Total protein derived from the astrocytes and NS/PCs was harvested with RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail (KeyGen Biotech Co., Ltd., Nanjing, China). The protein concentrations were measured using the Bradford method (Beyotime Institute of Biotechnology). The protein samples (50 μg) were fractionated by 10% SDS-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride (PVDF; Millipore, Billerica, MA, USA) membrane and immunoblotted with primary antibodies, including: rabbit anti-HMGB1 (1:1,000), rabbit anti-phosphorylated JNK (anti-p-JNK, 1:1,000; Cell Signaling Technology), rabbit anti-JNK (1:1,000; Cell Signaling Technology) and mouse anti-β-actin anbitody (1:1000, Beijing Ding Guo Changsheng Biotech) as an internal control. A Bio-Rad apparatus (Bio-Rad Laboratories, Richmond, CA, USA) and Quantity One software version 4.6.2 (Bio-Rad Laboratories) were used to scan the immunoblots for semi-quantitative analysis.

Cell pellets were lysed in the RIPA lysis buffer (KeyGEN, Nanjing, China) that contains 1% protease and phosphatase inhibitor cocktail (KeyGEN, Nanjing, China) for 30 min on ice. Then, lysates were centrifuged at 12,000×g for 15 min at 4 °C. The supernatants of the lysates were harvested for concentration determination and denatured at 100 °C. Denatured protein extracts measuring 40 μg were fractionated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Following blocking in TBST containing 5% bovine serum albumin for 1 h at room temperature, the membranes were incubated with primary antibodies (see Supplementary Table 2) diluted in Universal Antibody Diluent (NCM Biotech, Suzhou, China) overnight at 4 °C. On the second day, IRDye goat anti-mouse or rabbit secondary antibodies (Gene Company, Shanghai, China) were used to incubate the membranes at room temperature for 1 h. An LI-COR Odyssey infrared imaging system (LI-COR Biosciences, USA) was used for the visualization of protein blots. The Gray analysis of the target protein bands was performed with ImageJ and all protein expression levels were normalized to the GAPDH protein level of each sample. Comparisons were then made with the corresponding controls.