Rabbit anti enos

After lysing cells with extraction buffer, cell extracts were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. The following primary antibodies were used: rabbit anti-eNOS, KDR, Cyr61, Myl9, MMP9, ANGPTL3, and VEGF (abcam); EPHB4, RXRα, ERα, pAKT, AKT, pY925 FAK, FAK, pERK, ERK, and mouse anti-GAPDH (Santa Cruz Biotechnology). Antibody incubations were performed overnight at 4 °C. The secondary antibodies used were IRDyeTM-800-conjugated anti-mouse and anti-rabbit IgG (Li-COR Biosciences). Immunoreactivity was detected using an Odyssey Infrared Imaging System (Gene Company Limited). All immunoblots were repeated at least two–three times.

For detection of vWF, 2µl of plasma was electrophoresed and probed with sheep anti-vWF 1:1000; Abcam, MA, USA). Western blotting was carried out as published (26 (link)). For VCAM-1, a total of 40µg of protein from liver and gut lysates was resolved in 10% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad Laboratories Pty Ltd, Australia) and rabbit anti-VCAM-1 (1:1000; Abcam, USA) and rabbit anti-eNOS (1:500; Abcam, USA) was used. For plasma von Willebrand Factor (vWF) analyses, 2µl of sample were resolved in 7.5% polyacrylamide gels, and sheep anti-vWF 1:1000; Abcam, MA, USA) was used to detect vWF. A standard curve was created using known concentrations of recombinant vWF assessed via western blot, and the amount of vWF was calculated from that curve.