P22phox

Liver-extracted proteins (40 μg) were mixed with sample buffer, boiled for 5 minutes, and subjected to 8%, 10%, and 12% SDS-PAGE gel graded from maximum to minimum of protein molecular weight (120 volts, 60–90 min). Separated proteins on the gel were transferred to 0.45 μm polyvinylidene fluoride membranes. The membranes were then blocked with 5% fat-free dry milk in TBST at room temperature for 1 hour, followed by incubation overnight with antibodies (p47^phox, p22^phox, and GAPDH) (Abcam, Hong Kong, China) (diluted 1 : 500, 1 : 1000, and 1 : 10000) at 4°C. The next day, after being washed with TBST three times, the membranes were incubated with HRP-goat anti-rabbit antibody (KPL Company, Hong Kong, China) diluted 1 : 10000 at room temperature for 30 minutes. Then, after being washed with TBST four times, immunoreactive proteins were detected using the chemiluminescence method (LiDE110, Canon, Tokyo, Japan). Finally, band densities were determined using AlphaEaseFC software and quantified as the ratio between the OD value of the target band to that of GAPDH.

HUVECs were purchased from American Type Culture Collection (Rockville, MD, USA). Nitric Oxide Synthase Assay Kit, DHE, and DCFH-DA were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Antibodies against eNOS, p-eNOS, and iNOS were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against nitrotyrosine, NOX-2, p22^phox, p47^phox, and goat anti-rabbit IgG H&L (Alexa Fluor 488) were purchased from Abcam (Cambridge, MA, USA). Antibody against salusin-β was obtained from Bachem (Bubendorf, Switzerland). Antibodies against PPARγ, GAPDH, IL-1β, MCP-1, TNF-α, and VCAM-1 and HRP-conjugated secondary antibodies were purchased from Proteintech Group Inc. (Wuhan, China). 2-Chloro-5-nitro-N-4-pyridinyl-benzamide (T0070907) was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). The specific primers were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China).