Protein Expression Analysis in Kidney Samples

As previously described^{18 (link)}, kidney samples were run on polyacrylamide minigels. After transfer by electroelution to polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK), blots were blocked with 5% nonfat dry milk in Tris-buffered saline. Blots were then incubated overnight with antibodies against caspase 3 (1:500), heme oxygenase-1 (HO-1, 1:1,000), peroxiredoxin 6 (Prdx6, 1:1,000), and nitrotyrosine (1:1,000), all of which were obtained from Abcam (Cambridge, UK); and against glutathione peroxidase 4 (Gpx4, 1:1,000), obtained from Cell Signaling Technology (Danvers, MA, USA). We visualized the labeling with a horseradish peroxidase-conjugated secondary antibody, using enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA). We scanned the films with an imaging system (Alliance 4.2; UVItec, Cambridge, UK), after which we used densitometry to perform a quantitative analysis of the antibodies employed, normalizing the bands to β-actin expression.