Ab184166

Equal amounts of 200 µg of kidney cortical proteins were loaded per lane onto a polyacrylamide gel and subjected to electrophoresis. Following separation, the proteins were transferred onto nitrocellulose membranes. Membranes underwent treatment with a 0.1% Ponceau S solution (Sigma-Aldrich, St. Louis, MO, USA) for 10 min on a shaker, which was followed by rinsing with distilled water to eliminate background staining. Ponceau S staining served for total protein normalization.
The transferred proteins were then probed using specific antibodies, including a mouse eNOS antibody (1:250; BD610297BD, Biosciences, San Jose, CA, USA), a mouse nNOS antibody (1:200; SC-5302, Santa Cruz, CA, USA), a mouse DDAH1 antibody (1:500; SC-271337, Santa Cruz), or a rabbit DDAH2 antibody (1:2000; Ab184166, Abcam, Cambridge, UK). Subsequent to washing, the blots were incubated with the corresponding secondary antibody conjugated to horseradish peroxidase. Immunopositive bands were scanned using an imaging densitometer (Quantity One, Bio-Rad, Hercules, CA, USA) to quantify integrated optical density (IOD). Protein abundance was expressed as IOD normalized by Ponceau S stain (PonS, representing the total protein loaded). Complete blots and corresponding images of Ponceau S staining can be found in Supplementary Material.

CTX and dihydroethidium (DHE) were obtained from MedChemExpress LLC (HY-P1902A, Princeton, NJ, USA) and Sigma (D7008, St. Louis, MO, USA), respectively. Wheat germ agglutinin (WGA) was purchased from Biotium Inc. (29022, Fremont, CA, USA). The ADMA ELISA kit and TUNEL staining kit were purchased from Bio-Techne Co., Ltd. (NBP2–66728, Minneapolis, MN, USA) and Beyotime Institute of Biotechnology (C1090, Shanghai, China), respectively. Lactate dehydrogenase (LDH) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (A020-2-2, Nanjing, China). Antibodies against DDAH1, peroxiredoxin 3 (PRDX3), PRDX5 and β-tubulin were purchased from Signalway Antibody LLC (37368, 38567, 38828, 48659, Greenbelt, MD, USA). Antibodies against Bcl-2, Bax, DDAH2, Interleukin-6 (IL-6), M-cadherin and TNFα were purchased from Abcam (ab194583, ab182733, ab184166, ab259341, ab129078 and ab183218, Cambridge, UK). Antibodies against F4/80 and iNOS were purchased from Wuhan Sanying Biology Technology Company (28463-1-AP and 22226-1-AP, Wuhan, China). Antibodies against MyoD and myogenin were obtained from Santa Cruz Biotechnology, Inc (sc-32758 and sc-12732, Dallas, TX, UA).