Gel pro analyzer v 4

Total protein was extracted using RIPA lysis buffer (Cowin Bio, Beijing, China), and the concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of proteolytic products were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred to a nitrocellulose membrane (Merck Millipore, Heidelberg, Germany). After blocking with buffer (Beyotime, Shanghai, China) for 1 h, the membrane was incubated overnight at 4 °C with the following various antibodies: JNK (1:1000; Cat. No. 66210-1-AP), phospho-JNK (1:1000; Cat. No. 80024-1-AP), ERK1/2 (1:1000; Cat. No. 11257-1-AP), phospho-ERK1/2 (1:1000; Cat. No. 28733-1-AP), P38 (1:1000; Cat. No. 14064-1-AP), phospho-P38 (1:1000; Cat. No. 28796-1-AP; all from Proteintech, Wuhan, China), and β-actin (1:1000; Cat. No. 4970; Cell Signaling Technology, Danvers, MA, USA). Horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG and anti-mouse IgG; Beyotime, Shanghai, China) were used. Positive bands were detected via enhanced chemiluminescence (ECL; Tanon, Shanghai, China). The band intensity was analyzed semiquantitatively using Gel-Pro Analyzer v. 4.0 (Meyer Instruments, Houston, TX, USA), and the relative protein expression levels were normalized to match that of β-actin.

Cerebral tissues of the right hemisphere were homogenized in RIPA lysis buffer, and then centrifuged for 20 min at 4 °C, 12,000× g to collect the supernatant. Protein concentrations in lysates were determined with a BCA protein assay kit. Total protein at equal concentrations were separated by SDS-PAGE, and then transferred onto a PVDF membrane (Millipore). After blocking with 5% skimmed milk at room temperature, target proteins were probed with primary antibodies against p-p38, p-p65, p-IκB, GFAP, Iba-1, MMP-9, occludin, claudin 5, ZO-1, ICAM-1, VCAM-1, iNOS, IL-1β and β-actin (internal control) at 4 °C overnight. The following day, the membrane was incubated with the secondary antibody conjugated with horseradish peroxidase at room temperature and detected using an ECL plus kit. Membranes were imaged using Azure c300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, Dublin, CA, USA), and assessed using image analyzing software (Gel-Pro analyzer v4.0, Meyer Instruments, Houston, TX, USA). Results were expressed as the relative intensity of the target protein normalized to β-actin (as the internal control) in the cerebral tissues.