Figure 5.

Functional PDI is required for sufficient induction of apoptotic PERK signaling and is kept in a reduced state by ERp57. Every experiment was repeated twice and within the experiments at least three samples were treated in the same way. Bars represent mean±S.D. (a) Caspase-3 activity assay of shERp57 cells 96 h after KD induction and 24 h incubation with the PDI inhibitor 16F16. (b) HCT116 shERp57 cells were treated with various concentrations of the PDI inhibitor PACMA31 for 24 h after 72 h of KD induction and were used for caspase-3 activity assay. (c) Corresponding western blots of shERp57 cells. (d) At 96 h after ERp57 KD induction, nonreducing SDS-PAGE and subsequent western blot was performed with whole-cell lysates. (e) Redox-dependent mobility shift of PDI after mPEG-mal alkylation. At 96 h after ERp57 KD induction, shERp57 cells were subjected to the mPEG-mal alkylation protocol followed by SDS-PAGE and western blot. Oxidized cysteines of PDI were alkylated with the mPEG-mal₅₀₀₀ polymere, leading to an increased shift in gel migration. DTT and diamide were used as controls for fully reduced (PDI_red) and fully oxidized (PDI_ox) forms of PDI