Anti icam 1 bbig i1

Mouse aortas were perfused with PBS followed by 10% formalin or 4% paraformaldehyde. Aortic root sections were embedded in OCT for frozen sectioning. Photos of aortic root sections were taken with an Axiovert 35 Zeiss microscope (Zeiss, Germany) equipped with an Axiocam CCl camera at × 25 magnification. En face stained aortas and light microscopy was obtained with a Nikon Stereo microscope equipped with a Spot Insight camera (Spot Imaging) at × 5 or × 10 magnification. Aortic root sections and en face preparations were stained with Oil Red-O in 60% isopropanol or 80% methanol. Aortic roots were additionally stained with haematoxylin and eosin or trichrome by the UMASS morphology core, with rat anti-mouse CD68 (1:200) (AbD Serotec, clone FA-11) and Cy3-smooth muscle actin (Sigma S6198) (1:200), or with anti-ICAM-1 BBIG-I1 (1:200) or VCAM-1 BBIG-V1 (1:200) (R&D systems) and rat anti-mouse CD31 (1:400) (BD) and mounted in Prolong Gold with DAPI (Life Technologies). Images were quantified using ImageJ or Image Pro Plus Analysis Software (Media Cybernetics).

Cells were lysed in RIPA lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate and 50 mM EDTA) with 1 × HALT protease and phosphatase inhibitors (Thermo Scientific). Lysates were run on SDS–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. Membranes were immunoblotted with anti-ICAM-1 BBIG-I1 (1:1,000), VCAM-1 BBIG-V1 (1:1,000), E-selectin BBIG-E4 (1:1,000) (R&D systems), MAP4K4 A301-503A (1:2,000) (Bethyl), VE-cadherin 2500 (1:2,000), p-JNK1/2 4668 (1:1,000), total JNK1/2 9258 (1:1,000), p-p38 MAPK 4631 (1:1,000), p-IκBα 9246 (1:1,000), IκBα 4814 (1:1,000), p-p65 3039 (1:1,000), p65 8242 (1:1,000), HGK 3485 (1:1,000), p-Erk 4370 (1:1,000) (Cell Signaling Technology), total p38 MAPK sc-535 (1:1,000), total Erk1 sc-93 (1:4,000) (Santa Cruz) or lamin-β1 ab16048 (1:1,000) (Abcam) antibodies. Uncropped western blot images are provided as Supplementary Figs 4–6.