Angpt2

Spinal cord tissues were collected and washed with PBS, placed at 4^∘ C, and homogenized using T 25 digital homogenizer (IKA, Seoul, Korea) in lysis buffer (1× RIPA lysis buffer) and then finally passed through a 31^1/2 gauge syringe needle and centrifuged at 14,000 rpm at 4^∘C for 15 min. Protein concentration was determined in supernatants using Bio-Rad DC Protein Assay (Hercules, CA, USA). Equal amounts (40 μg) of protein were separated electrophoretically by 10% SDS-PAGE electrophoresis, and the resolved proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were incubated for 1 h with 5% non-fat skim milk prepared in TBS buffer to block nonspecific binding. The membranes were then incubated overnight with primary antibodies to ANGPT-1 (1:10000, Abcam, Cambridge, UK), ANGPT-2 (1:1000, Abcam), NE (1;1000), ZO-1 (1:500, Invitrogen, California, USA), AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), pAKT (1:1000, Cell Signaling Technology,) LC3B (1:1000, Cell Signaling Technology,), and Actin (1:10000, ABM). After 1-h incubation with corresponding secondary antibodies, The blots were visualized with a PowerOpti-ECL (Animal Genetics Inc., Gainesville, FL, USA) detection system, according to the recommended procedure. Immunoreactivity was detected using the BIORAD ChemiDoc™ XRS.

Proteins were isolated from treated RAW 264.7 cells and mice aortas. Tissues were lysed in lysis buffer (100 mM Tris-Cl, pH 6.8, 4%(m/v) SDS, 20% (v/v) glycerol, 200 mM β-mercaptoethanol, 1 mM PMSF, and 1 g/ml aprotinin) and proteins were transferred to PVDF membranes, which were then incubated with primary antibodies for VEGF-A (1:500, Millipore), ANGPT-1 (1:500, Abcam), ANGPT-2 (1:1000, Abcam), and GAPDH (1:5000, Sigma-Aldrich, Louis, USA) overnight at 4°C. The membrane bands were visualized by use of chemiluminescence (Millipore) and quantified by densitometry.

To assess overall burden and distribution of atherosclerosis, en face lesion staining with Oil-Red O was performed as previously described [15 , 42 (link)]. Cross-sections of the aortic roots (predilection site of atherosclerosis) were stained with hematoxylin and eosin (H&E) following a standard protocol of our lab. The content of lipids and collagen of aortic plaques was detected by Oil-Red O staining and Picrosirius red staining, respectively. The immunohistochemical staining procedure was as previously described [15 , 42 (link)]. Targeted proteins were identified by the following antibodies against macrophages/monocytes antigen (MOMA-2, 1:150, AbD Serotec, Oxford, UK), α smooth muscle actin (α-SMA, 1:200, Abcam, Cambridge, UK), ANGPT-1 (1:100, Abcam), ANGPT-2 (1:200, Abcam), and VEGF-A (1:150, Millipore).
Histological and immunohistochemical staining was analyzed by using Image-Pro Plus 6.0 (IPP 6.0, Media Cybernetics, MD, USA). The en face analysis of aortas was performed as previously described [15 , 42 (link)]. Plaque sizes were analyzed by H&E staining of the cross-sectional aortic sinus. Lipid deposition and content of collagen, macrophages, SMCs and angiogenic factors were analyzed as the ratio of the positive staining area of targeted protein to total plaque area. Fibrous cap thickness was evaluated by Picrosirius red staining as described [38 (link), 42 (link)].