Thioredoxin Reductase Activity Assay

For TrxR2 activity, a commercially available insulin reduction assay was utilized (Cayman Chemical). Cells were harvested in assay buffer (0.1 M NaPO₄, 5 mM EDTA, 0.1% Triton X-100, 1% protease inhibitor cocktail (Sigma Aldrich), 1% phosphatase inhibitor cocktails 2 and 3 (Sigma Aldrich), and 0.1 mM PMSF) and rotated for 2 hours at 4°C. Lysates were then spun at 10,000 rpm for 15 minutes at 4°C. Supernatants were collected and protein concentration was determined by BCA. Reductase activity was measured in a 96-well plate according to manufacturer’s instructions. Absorbance was measured at 412nm using a Spectramax M5 microplate reader.