Insulin Reduction Assay.

The reductase activity of PDI was monitored by observing the aggregation of insulin.^{37, 38} rPDI and oPDI were either kept at 25°C or heated to 90°C for 10 min prior to mixing at 3 μM concentrations with 104 μM insulin (Sigma-Aldrich) in the absence or presence of 450 μM of the PDI inhibitor quercetin-3-rutinoside (Q3R, Sigma-Aldrich).³⁹ The total volume was 135 μL in a buffer of 100 mM potassium phosphate, 2 mM EDTA, and 1 mM DTT. The 650 nm optical density of samples in a 96-well plate was read every minute for 90 min using a BioTek Synergy 2 plate reader. For clarity, only end-point measurements are shown.