Di e gssg

PDI disulfide reduction activity was monitored in PDI assay buffer containing 0.1 M potassium phosphate buffer pH 7.0 and 2 mM EDTA by adding 10 ng recombinant human PDIA3 (Prospec, ENZ-474) or 2 μg MTEC lysate to 150 mM DiE-GSSG (Cayman, 11547) in the presence of 5 μM DTT. DiE-GSSG is comprised of two eosin molecules attached to oxidized glutathione, resulting in proximity quenching of the eosin molecules. Upon reduction of the -S-S- bond (i.e. PDIs, DTT), fluorescence emission of eosin increases dramatically [25] (link). The increase in fluorescence signal was monitored at 528 nm with excitation at 485 nm using a Synergy HTX plate reader (Biotek). The total reaction volume was 100 μL. For inhibition of PDIA3 the reaction mixture was incubated with LOC14 (Tocris, 5606) at the indicated concentrations for 30 min on ice prior to the addition of DiE-GSSG. IC50s were calculated using GraphPad Prism (version 7.0, GraphPad); briefly initial rates of fluorescence over time were determined over the first 10 min for indicated inhibitor concentrations, IC50s were then determined using three-parameter non-linear regression.