Anti tenascin c

Lysates of aorta or macrophages were prepared and then separated by 10% SDS‐PAGE. Briefly, cells were homogenized and lysed with RIPA lysis buffer. 40 μg protein per lane was separated by 12% SDS–PAGE and electroblotted onto a poly (vinylidene difluoride) membrane (GE Healthcare). Following that, non‐specific binding was blocked by incubating with 5% non‐fat milk in TBST buffer at room temperature for 1 hr. The transferred proteins were incubated overnight at 4°C with various primary antibodies in Tris‐buffered saline with Tween buffer (20 mM Tris‐HCl, 150 mM NaCl and 0.1% Tween 20) followed by incubation with horseradish peroxidase‐conjugated secondary antibodies for 1 hr. Primary antibodies including anti‐tenascin‐c (1:500; Santa Cruz Biotech), anti‐annexin II (1:500; Santa Cruz Biotech), anti‐Akt (1:500; Santa Cruz Biotech), anti‐p‐Akt (1:500; Santa Cruz Biotech), anti‐p65 (1:500; Santa Cruz Biotech), anti‐p‐p65 (1:600; Santa Cruz Biotech), anti‐ERK1/2 (1:800; Santa Cruz Biotech), anti‐p‐ERK1/2 (1:800; Santa Cruz Biotech), anti‐HIF‐1α (1:500; Santa Cruz Biotech) and anti‐HIF‐2α (1:600; Abcam, Cambridge, MA, USA) were employed. After extensive washes in Tris‐buffered saline with Tween buffer, the signals on the membrane were visualized by enhanced chemiluminescence (GE Healthcare).

The expression of tenascin‐c and annexin II protein in mice aorta was analysed by immunohistochemical staining 28. Briefly, arterial sections (5 μm thick) were fixed in 5% formaldehyde before dehydrated with different doses of ethanol. The sections were then adhered to poly‐l‐lysine glass slides, washed with phosphate‐buffered saline (PBS 0.01 M, pH 7.4) and then treated with hydrogen peroxide to inactivate endogenous peroxides. The samples were then blocked with 1% bovine serum albumin and incubated with anti‐tenascin‐c (1:200; Santa Cruz Biotech, Dallas, TX, USA) and anti‐annexin II (1:200; Santa Cruz Biotech) antibody for 12 hrs at 44°C. After washing with PBS for three times, the sections were incubated with secondary antibody for 10 min. at 37°C and then incubated with horseradish peroxidase labelled streptavidin solution. Diaminobenzidine (DAB) was used for stain, with incubation for 1–2 min. The cover slips were counterstained with haematoxylin and then examined under light microscopy.