5.4. Redox-western blot and antibodies

For Prx2, Prx3 and Prx4 cells were incubated with NEM (50 mM) and washed with PBS- NEM (100 mM). Cells were scraped in alkylation buffer (40 mM Hepes, 50 mM NaCl, 1 mM EGTA, Inhibitors, Catalase, 100 mM NEM) and 1% CHAPS (from Applichem) for solubilization. For Trx1 and Trx2 cells were scraped in 20% TCA followed by two acetone washing steps. Proteins were dissolved in EB buffer (10% SDS, 150 mM NaCl, 50 mM Hepes) and incubated with 15 mM AMS (from Life Technologies) for 3 h. Protein amount was determined by Lowry protein assay. Samples were substituted with sample buffer (8.5% glycerin, 2% SDS, 6.25% TRIS/HCl pH 6.8, 0.013% bromphenol blue) and separated on a non-reducing SDS-PAGE gel, followed by Western blot analysis and detection by antibodies. Primary antibodies against Prx2 (#LF-PA0091), Prx3 (#LF-PA0030), Prx4 (#LF-PA0009), Trx1 (#LF-PA0187) and Trx2 (#LF-PA0012) were diluted 1:1000 and were purchased from AbFrontier. The antibody against Nox4 was a gift from Ajay Shah from Kings College London and was diluted 1:1000. After incubation with first antibodies, membranes were analyzed with an infrared-based detection system, using fluorescent-dye-conjugated secondary antibodies from LI-COR biosciences.