Bca reagent

The western blotting was performed as Linshi reported methods and modification. RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) and extraction kits (ThermoFisher Scientific, Shanghai, China) were used to isolate the total proteins, a nuclear protein, and cytoplasmic proteins, respectively. The protein concentration was analyzed using a BCA reagent (CWBIO, Beijing, China) followed by SDS-PAGE and electrotransfer of separated protein lysates to nitrocellulose membranes (Millipore). After 1 h of blocking, the membranes were incubated with a primary antibody at 4°C for overnight, then incubated with the secondary antibody for 2 h at room temperature. Positive bands were measured by enhanced chemo-luminescence (Tanon, Shanghai, China). Gel-Pro Analyzer software version 4.0 (Media Cybernetics, Silver Spring, MD, USA) was used to analyse the densitometry. Antibodies against ZO-1 (ab96587), occludin (ab167161), claudin-1 (ab129119), and claudin-4 (ab15104) were obtained from Abcam (Cambridge, MA, USA). The antibody against ERK (4695S), P-ERK (4370S), and β-actin (4970S) were secured from CST (CST, MA, USA).

Proteins were extracted from the brain samples using a Proteins Extraction Kit (CWBIO, China) and quantified with BCA-Reagents (CWBIO, China). Proteins were separated by SDS–PAGE at 160 V on a 12% gel (CWBIO, China) for 1 h and then transferred to a 0.22 μm nitrocellulose filter membrane at 200 mA for 3 h. The membranes were incubated with autophagy related primary antibodies diluted as follows: anti-CST3 antibody (ab24327; Abcam, UK), anti-mTOR antibody (ab32028; Abcam, UK), anti-mTOR (phospho S2448) antibody (ab109268; Abcam, UK), anti-LC3 antibody (ab192890; Abcam, UK), anti-Beclin-1 antibody (3495, CST, USA) diluted 1:1,000, and anti-GAPDH antibody (ab181602; Abcam, UK) diluted 1:2,000. Membranes were then incubated with secondary antibodies diluted at 1:5,000. The membranes were washed and bands were visualized with enhanced chemiluminescence immunoblotting detection reagents (Thermo Fisher Scientific, USA). For autophagic protein, bafilomycin A1 was used at a final concentration of 200 nM and added during the last 3 h. Images were gotten by Image Lab Software (BIO-RAD, USA), and semiquantitative analysis was performed by normalizing the protein bands to the internal control GAPDH.