Segments of the obstructed intestinal loops were fixed in 4% paraformaldehyde
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1 Supplementary text Supplementary materials and methods Histopathological examination Segments of the obstructed intestinal loops were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin wax with proper orientation of the crypt to villus axis before sectioning. Sections of 5 μm thickness were deparaffinized with xylene and graded ethanol, stained with haematoxylin and eosin (H&E), and observed under a light microscope. The villus height and crypt depth in all tissues were measured using a scale built in the AxioVision Rel. 4.6 software of the microscope (Zeiss). Three to six well-oriented crypt-villus units per animal sample and six to seven animals per group were examined. The ratio of crypt to villus was calculated and determined for each mouse. TUNEL assay Tissue sections and cell cultures were fixed with 4% PFA, and in situ detection of cells with DNA-strand breaks was performed by the TUNEL labeling method using a TdT-FragEL DNA fragmentation detection kit (Calbiochem) according to the manufacturer's instructions. Negative controls were performed by substituting tris-buffered saline (TBS) for the TdT enzyme.
2 Immunofluorescent staining of inos, phosphorylated MLC (pmlc), and phosphorylated PKCzeta (ppkczeta) in intestinal tissues Histological slides of intestinal tissues were deparaffinized with xylene and graded ethanol, and stained with antibodies against inos, pmlc, and ppkczeta. After quenching with 50 mm NH 4 Cl (Sigma) in PBS for 10 minutes at room temperature, tissues were blocked with 2% BSA for 2 hours at room temperature. Tissue sections were incubated with primary antibodies including rabbit anti-inos antibody (1:50, BD Biosciences), rabbit anti-phospho-myosin light chain 2 (Ser19) antibody (1:50, Cell signaling), and rabbit anti-phospho-pkczeta antibody (1:50, Cell signaling) overnight at 4 C. Some sections were incubated with rabbit IgG 1 isotype antibody in place of the primary antibody. The sections were washed three times with PBS and incubated with a secondary goat anti-rabbit IgG conjugated to Alexa Fluor 488 for one hour at room temperature. The tissues were then incubated with a Hoechst dye (1 μg/ml in PBS) (Sigma) for another 30 minutes. The slides were observed under a fluorescent microscope and the images were captured. ELISA Scraped mucosa was homogenized and sonicated in PBS, and the lysate was
3 centrifuged. The protein concentration in the supernatant was quantified. The levels of IFNγ in intestinal mucosa were measured using ELISA development kits (ebioscience) according to the manufacturer's instructions. The cytokine levels in intestinal mucosa were expressed in pg/mg of protein. Extraction of NP-40 soluble protein samples Caco-2 cells or scraped mucosa from mouse intestines were lysed with complete RIPA buffer for 60 minutes on ice. The lysates were centrifuged at g for 15 minutes and supernatant collected for protein assay. Protein samples were adjusted to 5 mg/ml, dissolved and boiled in 2X electrophoresis sample buffer. The samples were frozen at 20 C until used for western blotting. Extraction of cytosolic and membranous proteins in cells Cytosolic and membrane-bound extracts were prepared as described previously (41) by selective permeabilization with 0.05% digitonin (cytosolic lysis buffer) and 0.1% TX-100 (membranous lysis buffer). Briefly, cell monolayers were incubated with cytosolic lysis buffer for 15 minutes with gentle shaking, and the buffer containing the cytosolic proteins was collected. The remaining monolayer was incubated with the membranous lysis buffer for 10 minutes on ice, and the resultant
4 cell lysate was collected. Both cytosolic solution and cell lysate were centrifuged at g for 3 minutes, and the protein concentration of the supernatant from both fractions was quantified and adjusted to 0.1 mg/ml for cytosolic proteins and 4 mg/ml for membranous proteins. The samples were dissolved in 2X electrophoresis sample buffer and boiled at 95 C for 5 minutes. The samples were frozen at 20 C until used for western blotting. Western blotting Extracted protein samples were subjected to SDS/PAGE (4 13% polyacrylamide). The resolved proteins were electrotransferred onto PVDF or nitrocellulose membranes in a semi-dry blotter. Blots were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline (TBS) or 5% (w/v) bovine serum albumin in TBS with Tween 20 (TBS-T) for one hour at room temperature, washed three times with TBS-T (0.1% (v/v) Tween-20 in TBS), and incubated with primary antibody at 4 C overnight with gentle shaking. The membrane was washed with TBS-T and incubated with a secondary antibody for one hour at room temperature and then washed with TBS-T. The membranes were incubated with chemiluminescent solution (ECL, Millipore) for 1 minute with gentle agitation. Signal was detected on an UVP AutoChemi system (UVP, Upland). Band density was determined using the software Gel-pro Analyzer 4.0
5 (Media Cybernetics). The primary antibodies used for western blotting included anti-pkczeta (1:500, Cell Signaling), anti-ppkczeta (Thr410/403, 1:500, Cell Signaling), anti-mlc (1:500, Cell Signaling), anti-pmlc (1:500, Cell Signaling), anti-mek-1 (1:500, Santa Cruz), anti-zo-1 (1:500, Zymed), anti-occludin (1:3000, Zymed), anti-mypt1 (1:500, BD Biosciences), anti-pmypt1 (1:500, Cell Signaling), anti-inos (1:1000, BD Biosciences), anti-rock (1:1000, Cell Signaling), and anti-β-actin (1:10000, Sigma). The secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000, Santa Cruz) and goat anti-rabbit IgG (1:1000, Cell Signaling). Immunofluorescent staining and confocal microscopy Caco-2 cells were fixed with 4% PFA for 30 minutes, permeablized with 0.5% TX-100 for 5 minutes and quenched with 50 mm NH 4 Cl for 10 minutes. After blocking with 1% BSA for 10 minutes, cells were incubated with primary antibodies, including anti-zo-1 (1:100, Zymed), anti-occludin (1:200, Zymed) and anti-ppkczeta (1:50, Epitomics) for one hour at room temperature. Goat anti-rabbit or anti-mouse IgG conjugated to Alexa Fluor 488 were used as secondary antibodies. F-actin was labeled with phalloidin conjugated with Alexa Fluor 633 (Molecular
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