Enos 610297

Western Blot analysis was performed based on previous studies(14 (link), 15 (link)). Approximately half of the bladders were homogenized in RIPA with a protease inhibitor cocktail (Cell Signaling Technology, MA, USA). After centrifugation, total protein quantification was performed by the bicinchoninic acid method (BCA). Equal amounts(40 μg) of protein were fractioned on 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes for 1h at 100V. The membrane was incubated with blocking solution and then probed with 1:1000 diluted primary antibodies, including endothelial and neuronal NOS (eNOS; 610297 and nNOS; 610308) [BD Transduction Labs, CA, USA], inducible NOS (iNOS; sc-7271), CSE (sc-374249), CBS (sc-133154) [Santa Cruz Biotechnology, Dallas, TX, USA], 3-MST (NBP1-82617)[Novus Biologicals, Littleton, CO, USA], nuclear factor kappa B(NF-κB, 8242), hypoxia-inducible factor 1 alpha (HIF-1α, 36169), and β-actin (4970) [Cell Signaling Technology] at 4°C overnight. Following incubation with secondary antibodies, the visualization of protein bands was performed by a chemiluminescence substrate (Merck, Darmstadt, Germany) and Odyssey Fc system(LI-COR Biosciences, Lincoln, USA). The intensity of protein bands was quantified by Image J software(National Institutes of Health, Bethesda, Maryland, USA).