β actin

Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto PVDF membranes. Filters were probed with the following specific primary antibodies: anti-GCN5 (Santa-cruz), anti-AIB1 (BD Bioscience) anti-p21^Cip1/Waf1 (BD Biosciences), PCNA (Abcam, Cambridge, MA, USA), β-actin (Sigma, St Louis, MO, USA), Akt and phosphorylated-Akt (Cell signaling, Danvers, MA, USA), Anti-histone H3 (acetyl K9) antibody-ChIP Grade (Abcam, Cambridge, MA, USA) anti-CyclinD1 (Abcam, Cambridge, MA, USA). Blots were then incubated with horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL, USA) and visualized by chemiluminescence. The band density was quantified by densitometry using Scion Image software and normalized to β-actin levels.

IECs were lysed in buffer containing 0.1 M Tris-HCl (pH 7.5)/2% SDS/10% glycerol/5% 2-mercaptoethanol, boiled at 95°C for 5 min and centrifuged at 15000 rpm for 5 min and analyzed by reducing SDS-PAGE. Immunoblots were performed using antibodies against Claudin-1 (Abcam, Cambridge, UK, ab15098, 1∶100), Occludin (Applied Biosystems, 71–1500, 1∶250), Clock (Abcam, ab3517, 1∶200), Bmal1 (Abcam, ab3350, 1∶200) and β-actin (Santa Cruz, Texas, sc-1615, 1∶200). Quantitative analysis of Western blotting was done by using the Scion Image Software package and relative intensities of the target proteins to β-actin were shown.

One day after behavioral testing, six rats per group were decapitated for western blot analysis. DG regions were carefully dissected and immediately homogenized in lysis buffer with a cocktail of protease inhibitors. Protein concentrations were measured using the BCA assay kit (Beyotime, China). Equal amounts of protein from each sample were electrophoretically separated on 12% SDS-PAGE gels and transferred to PVDF membranes for analysis. The primary antibodies used were polyclonal rabbit anti-CD45 (1 : 1000, Abcam, USA), anti-CD11b (1 : 1000, Abcam, USA), and anti-β-actin (1 : 8000, Santa Cruz Biotechnology). The secondary antibody was horseradish peroxidase-conjugated to mouse anti-rabbit/mouse IgG (1 : 5000, Santa Cruz Biotechnology). The highly sensitive enhanced chemiluminescence kit, ECL (GE Healthcare, Buckinghamshire, UK), was used for these determinations. Protein band densities were quantified using the ImageJ software (NIH, Scion Corporation, Frederick, MD) and were normalized to β-actin. Final data were expressed as a percent of controls.