Lf pa0030

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.

To isolate proteins, samples were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS (Sigma Aldrich, L3771), 1% NP-40 (Sigma Aldrich, NP-40S), 0.25% sodium deoxycholate (Sigma Aldrich, D6750), 1 mM EDTA, 1 mM sodium fluoride, 1 mM Na₃VO₄) with a protease inhibitor cocktail (Roche Life Science, 11 697 498 001). For immunoblot analysis, proteins were electrophoresed on SDS-polyacrylamide gels and transferred onto PVDF membranes. The membranes were blocked with 4% skim milk (BD Difco, 232100) in Tris-buffered saline (TBS; 50 mM Tris-Cl, 150 mM NaCl, pH 7.5) containing 0.5% Tween-20 (Sigma Aldrich, P1379; TBST) and subsequently incubated with anti-PRDX1 (Abcam, ab41906), anti-PRDX2 (AbFrontier, LF-PA0007), anti-PRDX3 (AbFrontier, LF-PA0030), anti-PRDX4 (AbFrontier, LF-PA0009), anti-NR1H3/LXRα (Abcam, ab41902), anti-LC3B (Cell Signaling Technology, 2775), anti-SQSTM1/p62 (Abcam, ab56416), anti-CD36 (Santa Cruz Biotechnology, sc-9154), anti-LMNB/laminB (Santa Cruz Biotechnology, sc-6217) and anti-GAPDH (Santa Cruz Biothechnology, sc-25778) primary antibody and horseradish peroxidase-conjugated secondary antibody (Millipore, AP106P, AP124P and AP124P). Immunoreactive bands were detected with ECL™ western blotting reagents (GE Healthcare Life Sciences, RPN2106).

Cells treated with Hg compounds were probed for the oxidation state of peroxiredoxin 3 (Prx3) following the method described by Brown et al. [34] (link). After collection, cells were resuspended for 15 min in NEM lysis buffer consisting of 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 100 mM N-ethylmaleimide (NEM) and protease inhibitor cocktail in 40 mM HEPES, pH 7.4. Afterward, 1% CHAPS was added to samples to lyse cells and supernatants were collected after centrifugation for 4 min at 15,000g. Forty μg of the soluble protein fraction were separated by SDS-PAGE electrophoresis on a 4–12% Bis-Tris Mini gel in non-reducing conditions, transferred to a nitrocellulose membrane, blocked in 5% skimmed milk and probed with anti-Prx 3 rabbit polyclonal antibody (LF-PA0030, Ab Frontier) and goat anti rabbit secondary antibody. The ratio between the bands of Prx3 dimer and monomer was quantified for each sample using the Bio-Rad Quantity One software.