P c fos

Western blotting was performed using whole cell lysates. Aliquots of total protein (20-50 μg per lane) were electrophoresed on a 10% SDS-polyacrylamide gradient gel and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were incubated at 4 °C overnight with anti-GAPDH, MYC, c-Jun, p-c-Jun, c-Fos, p-c-Fos, JNK, p-JNK, or Igκ monoclonal antibody (all purchased from Abcam, Cambridge, MA). After rinsing in buffer wash, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX) diluted 1:10,000-30,000, followed by development with enhanced chemiluminescence reagents (Amersham, Little Chalfont, UK).

After 72 h, the HSFs were washed with PBS and lysed in lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) supplemented with proteinase and phosphatase inhibitor cocktails on ice. The lysates were collected after centrifugation at 12,000g for 30 minutes at 4°C. The BCA protein quantification kit (ThermoFisher, USA) was used to quantify the total protein of the extracted cells. 20 μg sample protein from each group was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane and blocked with 5% skimmed milk powder blocking solution at room temperature for 1 h. Then, the membranes were incubated at 4°C overnight with the antibodies for α-SMA (Immunoway, USA), Smad3 (Abcam, USA), p-Smad3 (Abcam, USA), c-FOS (Abcam, USA), p-c-FOS (Abcam, USA),c-Jun (Abcam, USA), p-c-Jun (Abcam, USA), and GAPDH (Abcam, USA). After washed with TBS, the membranes were added secondary antibody (1 : 10 000) and shook gently at room temperature for 40 minutes, and ECL was added to the membrane to react for 3–5 minutes, and the film was exposed. The density of the bands was normalized to β-actin and quantified with Image software. Immunofluorescence was assessed using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

The 2-Chloroethanol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Ningbo, China). Reagents for the primary culture of astrocytes were purchased from Biological Industries (Beit-Haemek, Israel). The quantitative real-time (RT)-PCR assay kit was purchased from Takara, Japan. The enhanced chemiluminescence (ECL) plus kit, bicinchoninic acid (BCA) protein assay kit and NE-PER™ nuclear and cytoplasmic extraction reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). SB202190, pyrrolidine dithiocarbamate (PDTC), and SR11302 were purchased from Selleck (Houston, TX, USA) and APExBIO (Houston, TX, USA). Primary antibodies against MMP-9, p65, IκBα, c-Jun, c-Fos, p-c-Jun, p-c-Fos, p-IκBα, and LaminB were products of Abcam (Cambridge, UK) and Cell Signaling Technology (Beverly, MA, USA). Antibodies against glial fibrillary acid protein (GFAP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β-actin were obtained from Millipore (Billerica, MA, USA), Proteintech (Wuhan, China), and ABclonal (Wuhan, China), respectively. The secondary antibodies conjugated with Alexa Fluor 488 or tetramethylrhodamine (TRITC), RIPA Lysis Buffer, and DAPI were obtained from Beyotime Biotechnology (Shanghai, China).