Sc 271337

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.

The lung tissues or cells were collected and homogenized in RIPA lysis buffer (Roche Applied Science, Indianapolis, IN). The protein concentration was tested using a BCA protein assay kit (Beyotime, China). Then, 15 μg/lane samples were loaded onto a 4–20% gel, resolved by SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). The membranes were blockaded with 5% skimmed milk for 2 h to block non-specific binding. The PVDF membranes were mixed with primary antibodies and incubated at 4°C overnight. The next day, after rinsing with TBST, the membranes were cultured with the HRP-conjugated secondary antibody (Abcam, ab6789, 1:2,000) for 2 h at room temperature. The band density was tested using Quantity One software with an ECL kit (BioRad Laboratories, Shanghai, China). The primary antibodies used were: p-eNOS (ser1177) (PA5-35879, 1:500, Thermo Fisher Scientific, USA), DDAH1 (sc-271337, 1:1,000, Santa Cruz Biotechnology, Inc., USA), and β-actin (ab8226, 1:1,000, Abcam) served as loading control.