Labworks

In order to prepare the protein sample, the frozen jejunum tissue was lysed using tissue protein lysis buffer, and the protein concentration was determined according to the instructions of the bicinchoninic acid (BCA) protein quantitative kit (Sun Bio, Beijing, China). Sodium dodecyl sulfate–polyacrylamide gels (10–12%) were configured according to the weight of detected proteins. Fifty micrograms of protein in each sample was extracted to perform electrophoresis, transferred to nitrocellulose filter membranes, then was incubated overnight at 4°C with primary antibodies against occludin (1:1,000, ab216327), ZO-1 (1:1,000, ab96587), Rho (1:1,000, ab40673), ROCK1 (1:2,000, ab45171), and β-actin (1:2,000) (all from Abcam, Cambridge, MA, USA). After being washed three times, the membranes were incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc. USA) for 2 h at 37°C. Then the membranes were reacted using enhanced chemiluminescence (ECL) solution (Millipore, Corporation, Billerica, MA, USA), and then were scanned (Konica Minolta Medical Imaging, Inc., Wayne, NJ, USA). The expression of protein was quantitatively analyzed using target protein/β-actin with Adobe Photoshop (Adobe, Mountain View, CA, USA) and Lab Works (UVP, Upland, CA, USA) software.

The HSC-3 and HOEC cells were lysed in RIPA buffer, and total protein was quantified using bovine serum albumin (BSA, Boster, Wuhan, China). After being added with 5× loading buffer and boiled for 5 min, the proteins were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, U.S.A.). The membranes were blocked with 1× Blotto and incubated with primary antibodies (anti-KRT84 antibody, 1:1000, Abcam, U.S.A.; anti-GAPDH antibody, 1:1000, Abcam, U.S.A.) at 4°C overnight. After incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (abcam, U.S.A.) at room temperature for 1.5 h, the membranes were developed with Western Lightning™ Chemiluminescence Reagent (PerkinElmer, U.S.A.). The intensity of protein was measured by using LabWorks™ (UVP, U.S.A.), with GAPDH as the internal control.

Proteins (30 μg) from each sample were subjected to SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) (10% acrylamide, w/v) and transferred to PVDF (polyvinylidene fluoride) membranes (Amersham Biosciences, Uppsala, Sweden). Nonspecific binding sites were blocked with 5% (v/v) nonfat milk in 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 0.1% (w/v) Triton X-100 at 20°C at room temperature for 1 h. Immunological detection was performed using the anti-MMP9 antibody at 1:1000 dilution (Cell Signaling Technology, Inc, Danvers, MA, USA), anti-S100A9 at 1:250 dilution (HPA004193, Sigma-Aldrich), and anti-clusterin at 1:100 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing three times, blots were further incubated with a horseradish peroxidase-conjugated secondary antibody (1: 5000 dilution) at room temperature for 1 h. Protein intensities were determined using densitometry (LabWorks, UVP, Inc., Upland, CA, USA). Differences with p values<0.05 were considered to be statistically significant.