Comparison of purified ERp57, PDI, and ERp5 and characterization of rabbit anti-ERp57 Abs. (A) The relative sizes of the 3 enzymes on a gel under reducing conditions. The enzymes were expressed in E coli and purified by the His tag using a nickel column. (B) The enzymatic activities in the GSSG assay. A total of 150nM Di-E-GSSG was incubated in the presence of ERp57, PDI, or ERp5. The amount of EGSH formed over time is shown. (C) The reactivity of rabbit Ab 1 (Ab1) and Ab 2 (Ab2) on Western blot of platelet lysate (4 × 108 platelets/lane) and purified ERp57 (lane 1) or PDI (lane 2; 200 ng protein/lane). (D) The inhibitory effect of Ab1 and Ab2 against ERp57 in the Di-E-GSSG assay compared with a normal rabbit IgG control (nL IgG); 30 μg/mL of each Ab was used in these studies. For the curves in panels B and D, the enzyme was added at ∼ 60 seconds.
