Computational protocol: New Role for L Arginine in Regulation of Inducible Nitric Oxide Synthase Derived Superoxide Anion Production in Raw 264.7 Macrophages

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Protocol publication

[…] After the treatment procedure, the RAW 264.7 cells were lysed using SDS-lysing buffer. The same amount of protein (30 μg) from each lysate was subjected to SDS-polyacrylamide gel electrophoresis, as described previously []. After electrophoresis, the proteins were transferred to a PVDF (Immobilon-P) membrane and then incubated with a mouse iNOS-specific antibody (1/5000) (Anti-iNOS/NOS Type II mAb, Transduction Laboratories, USA) for 24 h, and with horseradish peroxidase-labelled anti-mouse IgG antibody (1/2000) (ECL Anti-mouse IgG, Biosciences, USA) for 1 h. The equal loading of proteins was verified by β-actin immunoblotting (1/5000, SantaCruz Biotechnology, USA). The blots were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, USA) and exposed to CP-B X-ray films (Agfa, Czech Republic). The relative levels of the proteins were quantified by scanning densitometry, using the ImageJ program, and the individual band density value was expressed in arbitrary units. [...] Data were statistically analyzed using a one-way analysis of variance (ANOVA), which was followed by Dunnett's multiple comparison test (Statistica for Windows 8.0, Statsoft, Tulsa, Okla, USA). All data are reported as means ± SEM. A P value of less than 0.05 was considered significant. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Chemicals NADP, Nitric Oxide, Superoxides, Peroxynitrous Acid