Ab76199

Western blotting was performed as previously described [20 ]. Proteins were collected using RIPA buffer (P0013B, Beyotime, China), protease inhibitors (p1005, Beyotime, China), and phenylmethylsulfonyl fluoride (ST505, Beyotime, China), quantified by a protein quantification kit (P0011, Beyotime, China), and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, electrophoresis was conducted to transfer the proteins into a PVDF membrane (160-0184, Bio-Rad, USA). The membrane was blocked by 5% dried skimmed milk powder for 1 h and then incubated with antibodies against Akt (1: 500, 56kDa, ab8805, Abcam, UK), phosphorylated (p)-Akt (1: 1000, 56kDa, ab38449, Abcam, UK), endothelial nitric oxide synthase (eNOS) (1: 1000, 133kDa, ab76198, Abcam, UK), p-eNOS (1: 500, 140kDa, ab76199, Abcam, UK), and GAPDH (1: 1000, 36kDa, ab8245, Abcam, UK). After incubation for 24 h, the membrane was washed by 1% Tris-buffered saline with Tween 20 and incubated with goat anti-rabbit secondary antibody (1: 10 000, ab205718, Abcam, UK) or goat anti-mouse secondary antibody (1: 10000, ab6789, Abcam, UK). An ECL luminescence kit (PE0010, Solarbio, China) and a gel imaging system (FluorChem FC3, Alpha, USA) were used to expose the membrane and visualize the protein bands, respectively. ImageJ2x (Rawak Software, Germany) was used to analyze the results.

The protein was extracted from the cells with the RIPA (Beyotime, P0013C). The protein concentration was determined using the bicinchoninic acid (BCA) method, and then the protein was incubated at 99°C for 10 minutes to denature the protein. After that the samples were separated by the 10% sodium dodecyl sulfate (SDS) gels (Beyotime, P0012A). Then the proteins were transferred to the PVDF (polyvinylidene fluoride) membranes (Millipore, USA). After that the targeted proteins were incubated with the primary anti-body p-eNOS (Abcam, ab76199), eNOS (Abcam, ab184154), endothelin-1 (ET-1) (Abcam, ab2786), Bax (CST, #14796), Bcl-2 (CST, #15071), cleaved caspase-3 (CST, #9664), caspase-3 (CST, #9662), α1-AMPK (Abcam, ab32047), Nox-2 (Abcam, ab80508), and GAPDH (CST, #5174) at 4°C overnight. In the second day, the membranes were washed by the PBST (phosphate-buffered saline Tris) for 3 times. After that, the membranes were incubated with the second anti-body rabbit IgG (CST, #14708) and anti-mouse IgG (CST, #7076) for 2 hours at the room temperature. Then the membranes were washed by the PBST again. Then the membranes were exposed in the machine.

Western blotting was performed as previously described (13 (link)). Briefly, protein was extracted from 1.5×10⁶ cells, and the following antibodies were used: Anti-endothelial nitric oxide synthase (eNOS; ab76198; 1:1,000; Abcam), anti-phosphorylated (p)-eNOS (ab76199; 1:1,000; Abcam), anti-GATA4 (ab84593; 1:1,000; Abcam), anti-NOX4 (ab133303; 1:1,000; Abcam) and anti-GAPDH (ab8345; 1:1,000; Abcam) primary antibodies, and HRP-conjugated secondary antibodies [goat anti-rabbit (ab6721; 1:2,000; Abcam), goat anti-mouse (ab6789; 1:2,000; Abcam;)]. Image J version 1.5.1 (National Institutes of Health, Bethesda, MD, USA) was used to perform densitometric analysis.