Xar film

Proteins were extracted from harvested cells, and their concentration was determined by the BCA assay (Pierce). Protein samples (30 μg) were resolved by SDS-PAGE and transferred to a PVDF membrane. The following antibodies were used: IL-22R1 (ABCAM, ab211675), Stat1 (Cell Signaling Technology, number 9172), Stat3 (Cell Signaling Technology, number 9139), p-Stat1 (Cell Signaling Technology, number 9167), and p-Stat3 (Cell Signaling Technology, number 9145). The results were visualized with a Kodak autoradiography film (Kodak XAR film).

Acrylamide gels for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were prepared as described by Sambrook and Russell (Sambrook and Russell 2001 ). The final acrylamide concentration was 12.5% with an acrylamide:bis-acrylamide ratio of 29:1. 30 μg of mitochondrial protein was loaded in one lane of a gel. Following electrophoresis proteins were transferred onto a Bio Rad Immun-Blot PVDF membrane (Bio Rad, Berkeley) as described (Towbin et al. 1979 ). Following transfer, the membrane was blocked for 1 hr at room temperature in blocking solution (5% fat-free skim milk powder in TBST (0.2 M Tris-HCl pH 7.5, 0.15 M NaCl, 0.5% Tween 20)) with gentle shaking. The blocking solution was removed and the membrane was incubated with gentle shaking for 1 hr with primary antibody at an appropriate dilution in blocking solution. The membrane was then washed three times, with gentle shaking for 5 min with TBST, followed by a 1 hr incubation with gentle shaking at room temperature with secondary antibody (1:3000 dilution of goat anti-rabbit antibody HRP-conjugate solution) in 50 ml blocking solution. The membrane was then washed three times for 10 min with TBST, and once with TBS (TBST without Tween 20). Detection of bound antibodies was facilitated using the LumiGLO chemiluminescent detection assay (KPL, Maryland) with Kodak XAR film (Eastman Kodak, New York).

The mesenteric tissue from each rat was washed in PBS, and the proteins were extracted in cold 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, and a protease inhibitor mixture (P2714, Sigma). After sonication for 15 s (3 times with 10-s intervals) and extraction for 30 min at 4°C, the sample extracts were clarified by centrifugation at 14,000×g for 20 min and used immediately or stored at –20°C. The proteins were separated on 10% polyacrylamide slab gels and transferred to 0.22-µm nitrocellulose membranes (GE, Germany). Nonspecific reactivity was blocked by incubation for 1 h at room temperature in 5% non-fat dry milk dissolved in washing buffer (PBS, pH 7.6, 0.2% Tween 20). The blots were incubated with anti-p65 and anti-VCAM-1 antibodies (0.2 µg/mL in blocking solution) for 60 min at room temperature. Horseradish peroxidase-conjugated goat anti-rabbit-IgG and swine anti-goat-IgG dissolved in blocking buffer were used as secondary antibodies (0.25 µg/mL, 45 min at room temperature). Excess first and second antibodies were removed by washing 5 times for 5 min in blocking solution. Detection was accomplished with an enhanced chemiluminescence system (ABC, Dako System) and subsequent exposure to Kodak X-AR film (Eastman Kodak) for 5–30 s.

Cultured FLS were washed with PBS, and protein was extracted with lysis buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl₂, 1.5 mM EDTA, 20 mM β-glycerophosphate, 50 mM NaF, 1 mM Na₃VO₄, 10 μg/ml aprotonin, 1 μM pepstatin A, 1 mM phenylmethylsulphonyl fluoride). The protein concentrations were determined with the DC protein assay kit (Bio-Rad, Hercules, CA, USA). Whole cell lysates containing 50 μg of protein were fractionated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline plus 0.1% Tween 20 (TBST) containing 5% non-fat milk for 1 h at room temperature followed by incubation overnight with the appropriate antibody at 4 °C. The membrane was washed three times and incubated with horse radish peroxidase-conjugated secondary antibody for 1 h. Immunoreactive protein was detected by chemiluminescence with Kodak X-AR film (Eastman Kodak, Rochester, NY, USA).

The expression of the following cytokines was assessed using the ChemiArray system (rat antibody arrays) (Chemicon International, USA): neutrophil chemotactic cytokine 2 and 3 (CINC-2 and CINC-3), CX3CL1, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-3 alpha (MIP-3 alpha), nerve growth factor beta (beta-NGF), tissue inhibitor of metalloproteinase-1 (TIMP-1), vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (INF-γ), interleukin 1 alpha and beta (IL-1α, IL-1β), interleukin 4, 6 and 10 (IL-4, IL-6, IL-10) LIX, leptin, tumor necrosis factor alpha (TNF-α). The procedures were carried out according to the manufacturer’s instructions. Detection was performed with a chemiluminescence system and subsequent exposure to Kodak X-AR film (Eastman Kodak) for 5–30 s. The cytokines were distributed in the membranes according the map (Table 1).