Epr4776

Staining was performed using a Discovery Ultra Autostainer (Ventana). For each immunohistochemistry (IHC) Ab, positive control tissue was initially used for optimization and identification of staining morphology, typically with archival paraffin-embedded tonsil, thymus, bone marrow, or spleen. Optimal Ag retrieval, primary Ab dilution, and detection kits were then determined by staining 4-µm sections of intestine. All Ag retrieval was optimal using a Standard Cell Conditioning 1 buffer (Ventana) retrieval protocol for 64 min at 100°C. The following anti-human Abs were optimized: anti-pSMAD3 (phosphor-S423 and S425, rabbit monoclonal clone EP823Y, dilution 1:100; Abcam), anti–MAdCAM-1 (mouse monoclonal, clone 355G8; Thermo Fisher Scientific, dilution 1:100), anti–ICAM-1 (rabbit monoclonal, clone EPR4776, dilution 1:400; Abcam), and anti–VCAM-1 (rabbit monoclonal, clone EPR5047, dilution 1:250; Abcam). The Ultraview DAB Detection Kit (Ventana) was used for all Abs. Digital images were acquired using a Vectra 3 Slide Scanner (PerkinElmer) for cell adhesion molecules and an BX43 microscope (Olympus) for pSMAD3 IHC. Unless otherwise noted, representative images were selected from 10 samples stained in each group.

HUVECs and the whole aorta were lysed in RAPA buffer (150 mM NaCl, 50 mM Tris, 0.1% SDS, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF and protease inhibitors). Proteins were quantified with a BCA assay. Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441). Western blots were analyzed using a Tonen Image System.