Occludin

Proteins were extracted from the small intestine by using a tissue lysis buffer (Thermo Scientific, Waltham, MA, USA) with a protease inhibitor cocktail (Roche Diagnostic, Manheim, Germany). Samples were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked by 5% dried skimmed milk for 1 h, and were incubated with the corresponding primary antibodies under rotation overnight at 4 °C. The membranes were incubated for 1 h with the peroxidase-conjugated secondary antibody at room temperature, and the proteins were visualized using a chemoluminescence system (Fusion, Paris, France) with Millipore Immobilon ECL substrate (Millipore, Inc., Burlington, MA, USA). The primary antibodies used were Occludin (1:1000, Abcam, Waltham, MA, USA), Claudin-1 (1:1000, Invitrogen, USA) and β-actin (1:1000, Santa Cruz, Dallas, TX, USA), respectively.

The ileum and colon tissues were homogenized in RIPA lysis buffer containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein homogenates were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After blocking for 1 h with 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween (TBST: 50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.4), the membranes were incubated overnight with specific primary antibodies against Occludin (Abcam, Cambridge, UK), ZO-1 (Zonula occludens) (Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin (Zsbio, Beijing, China) at 4 °C. Then, the membranes were incubated for 1 h with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit or anti-mouse IgG-HRP) (Jackson ImmunoResearch Inc., West Grove, PA, USA), and the bands were detected by using enhanced chemiluminescence. The blots were scanned by a Bio-Rad ChemiDoc XRS and the intensity of each protein was quantified by Gel Image system V4.00 software (Tanon, Shanghai, China).

The hBMECs grown in 12-well dishes were fixed with 4% paraformaldehyde for 15 min, followed by three washes with PBS. Cells were incubated with the primary rabbit ZO-1, Occludin, or Claudin-5 antibody (Abcam, Cambridge, MA, USA) overnight at 4 °C and then incubated with Cy3 goat anti-rabbit antibody (Proteintech, Chicago, IL, USA) for 1 h. Cells were counterstained with DAPI to visualize the nucleus morphology and mounted and photographed using the BX41 fluorescence microscopy (Olympus, Tokyo, Japan).

The freshly isolated cerebellum tissues were collected and homogenized. Protein content was determined with a BCA Protein Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, USA). A total of 10–30 μg protein was loaded in each lane and was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After separation, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Hertfordshire, UK). The membranes were blocked with 5% bovine serum albumin. Blots were incubated overnight with the primary antibodies as follows: occludin (1:1000, Abcam, Cambridge, MA, USA); ZO-1 (1:500, Invitrogen, Carlsbad, CA, USA); claudin-5 (1:500, Abclonal, Woburn, MA, USA); ICAM-1(1:1000, Abclonal, Woburn, MA, USA); VCAM-1 (1:1000, Abclonal, Woburn, MA, USA); Ang-1 (1:500, Abclonal, Woburn, MA, USA); Tie-2 (1:500, Abclonal, Woburn, MA, USA); GAPDH (1:5000, Abclonal, Woburn, MA, USA). Following 45–60 min incubation with horseradish peroxidase-conjugated rabbit or mouse secondary antibody (1:10,000, BioPM, Wuhan, China), blots was visualized using the enhanced chemiluminescence (ECL) detection system (Thermo Scientific, Waltham, MA, USA). Band densities were measured by densitometry and were quantified with Image J software (version 1.46r, Bethesda, MD, USA). GAPDH was used as a control.