Rabbit enos

Western blotting was performed as described previously [27 (link)]. Tissue and cell lysates were probed with the following antibodies at a concentration of 1:1000: mouse DJ-1 (sc#55572), mouse β actin (sc#69879), mouse PGC-1α (sc#517380), (Santa Cruz, CA, USA), rabbit HMOX-1 (#86806, Cell Signaling, Danvers MA), rabbit eNOS (#32027, Cell Signaling), rabbit iNOS (#ab178945, Abcam, Cambridge, MA, USA), rabbit p62 (#H0023636-P01, Abnova, Walnut, CA, USA), rabbit LC3 (#D3U4C, Cell Signaling), and rabbit Rubicon (#8465, Cell Signaling) for an hour at room temperature or overnight at 4 °C, and then incubated with horseradish peroxidase conjugated secondary antibodies for an hour, using Bio-Rad Gel Doc 2000. Densitometry was performed using Gel-Quant-Net software. Plasma DJ-1 was measured by enzyme-linked immunosorbent assay ( ELISA )(#DY3995, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Assay range:62.5–4000 pg/mL.

Rat aorta tissue and endothelial cells were collected and subjected to disruption using an ultrasonic homogenizer in RIPA containing protease and phosphatase inhibitors (Jiangsu KeyGEN BioTECH Corp., Ltd. Nanjing, China). Protein concentrations were detected using the Pierce BCA protein assay kit (Rockford, IL, USA). Twenty micrograms of protein from the cells (8 μL for animal tissue) were separated on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes, which was blocked using 5% nonfat milk for 1 h at room temperature. Protein samples were incubated overnight with antibodies, rabbit β-actin, and rabbit eNOS (Cell Signaling Technology, Beverly, MA, USA). The secondary antibody, antirabbit immunoglobulin G-horseradish peroxidase (1 : 5000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Signals were detected and analyzed using the Tanon-4500 Gel Imaging System (Tanon Science and Technology Co., Ltd., Shanghai, China).