Enhanced chemiluminescence reagent kit

Western blot was performed as previously described [31 (link)]. Briefly, the thoracic aortic tissues from all groups were quickly isolated and snap-frozen. Total proteins (for KOR and eNOS) were extracted with lysate and nuclear proteins (for NF-κB p65) were isolated using a nuclear NE-PER extract kit (Thermo Scientific, IL, USA). Equal weight of proteins (40 μg) were loaded into a SDS-PAGE gel. After separated, the proteins were transferred onto nitrocellulose membranes electrophoretically. Then the membranes were incubated with 5% skim milk for 1 hour, appropriate primary antibody at 4°C overnight, and secondary antibody at room temperature for 1 hour in order. Enhanced chemiluminescence reagent kit (Millipore) was used for development of the blots, which were detected with UVP Bio-Imaging Systems. Protein levels were evaluated by Vision Works LS Acquisition and Analysis Software.
The following primary antibodies were used: KOR (1:1 000), endothelial nitric oxide synthase (eNOS, 1:1 000), phospho-eNOS (at Ser-1177) (1:1 000), NF-κB p65 (1:1 000), histone 3 (1:1 000) and β-actin (1:1 000). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG and rabbit anti-goat IgG at 1:5,000 dilution. All above antibodies were purchased from Santa Cruz Biotechnology.

Protein samples isolated from cultured cells using the RIPA lysis buffer (Cat# P0013C, Beyotime) were electrophoresed on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking for 1 h with 5% skim milk, the membranes were incubated with specific primary antibodies at 4 °C overnight, followed by the secondary antibodies at 37 °C for 2 h. The immunoreactive signals were visualized by enhanced chemiluminescence reagent kit (Millipore). The primary antibodies used in this study: anti-PAK1 (1:1000, Cat#2602, Cell signaling, Boston, MA, USA), anti-CD63 (1:1000, ab134045, Abcam, Shanghai, China), anti-CD9 (1:2000, ab92726, Abcam), and anti-GAPDH (ab18602, 1: 5000, Abcam) [26 (link)].

Western blotting was performed to confirm the results of the integrated analysis. Briefly, protein concentrations were determined by a BCA Kit (Thermo, United States). Protein samples (20 μg) were separated by SDS-PAGE and transferred onto PVDF membrane (Millipore, United States). Then the membranes were blocked with 5% skim milk in TBS solution supplemented with 0.1% Tween-20 (TBST) for 1 h at room temperature and subsequently incubated overnight at 4°C with specific primary antibodies. After that, membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Finally, the protein bands were visualized by an enhanced chemiluminescence reagent kit (Millipore, MA). The specific information of antibodies used in this study is shown in Supplementary Table S2. Densitometry was quantitated using ImageJ software and normalized to GAPDH expression.