Image quant 300 cabinet

Total protein extracts were obtained after washing the hearts with PBS and adding 300 ml of RIPA modified lysis buffer (50 mM NaCl, 50 mM Tris–HCl (pH 7.40), 1% Triton X-100, 1 mM EDTA, 1 mM PMSF; 2.5 g/l Protease Inhibitor Cocktail (Sigma-Aldrich Co., St. Louis, MO, USA), 1 mM Na₃VO₄, 1 mM NaF), or washing the cultured cells and scraped off the dishes with 50 µl of the same buffer. Then, the tubes were kept on ice for 30 min with swirling, and the samples were centrifuged at 7,000 g at 4°C for 10 min. The supernatants were stored at −20°C. Protein concentrations were determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad, USA) and bovine serum albumin (Sigma-Aldrich Co., St. Louis, MO, USA) as a standard (36 (link)). For Western blot analysis, total proteins were boiled in Laemmli sample buffer, and equal amounts of protein (40–50 µg) were separated by 10–12% SDS-PAGE. The gels were blotted onto a Hybond-P membrane (GE Healthcare, Madrid, Spain) and incubated with the following antibodies: anti-NOS2, anti-NOS3, anti-Arginase I (Arg-I), anti-CD31, anti-VEGF-A, and anti-α-actin (Santa Cruz Biotechnology, CA, USA). The blots were revealed by enhanced chemiluminescence in an Image Quant 300 cabinet (GE Healthcare Biosciences, PA, USA) following the manufacturer’s instructions. Band intensity was analyzed using the NIH-ImageJ software (37 (link)).

Group settings were consistent with the description of nitric oxide production assay. Total protein was extracted after washing the PBMCs with PBS, and 70 μl of RIPA modified lysis buffer was added (Beyotime, Nanjing, China). Protein concentrations were determined through the Bradford method used by Bio-Rad Protein Assay (Bio-Rad, USA) reagent and bovine serum albumin (BSA, Sigma-Aldrich Co.) as a standard. Proteins (30 µg) were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) for Western blot analysis. The non-specific binding was blocked with 5% BSA. The membranes were cleaned three times via TBST and then incubated with first antibodies (anti-p (Tyr705)-STAT3 (Novus Biologicals, USA), anti-SOCS3 (Beyotime, Nanjing, China), anti-IL-10 (Affinity Biosciences, china), and anti-β-actin (AB clonal, China)) overnight at 4°C. The membranes were again washed three times and incubated with HRP-conjugated goat anti-rabbit IgG (Beyotime, China) at 37°C for 1 h. Finally, the blots were revealed by enhanced chemiluminescence (ECL) in an ImageQuant 300 cabinet (GE Healthcare Biosciences, USA) according to the manufacturer’s instructions. The band intensity was analyzed through ImageJ software.