A plasmid-encoding, human full-length TDP-43 was a kind gift from Dr. H.-N. Du (College of Life Sciences, Wuhan University). Recombinant, full-length wild-type human TDP-43 (residues 1-414) containing a tag of six histidine residues (polyhistidine tag) at its C-terminal domain was constructed into a prokaryotic expression vector pET22b and expressed in E. coli BL21 (DE3) cells (Novagen, Merck, Darmstadt, Germany). TDP-43 protein was purified to homogeneity by nickel affinity chromatography as described by Vega et al. (70) . After purification, the refolded TDP-43 protein was dialyzed against 50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl four times to remove the detergents and salts, concentrated, and then centrifuged at 17,000 g for 30 min at 4 °C to remove large aggregates. The supernatant of the refolded TDP-43 protein was freshly used or stored at -80 °C. SDS-PAGE was used to confirm that the purified human TDP-43 protein was single species. We used a BCA protein assay kit (Beyotime, P0012) to determine the concentration of human TDP-43 protein.
A plasmid-encoding, human full-length PDI was a kind gift from Dr. L. W. Ruddock (Faculty of Biochemistry and Molecular Medicine, University of Oulu). The gene for PDI 1-491 was constructed in a prokaryotic expression vector pET23, and a PDI mutant dnPDI was constructed by site-directed mutagenesis using a wild-type PDI template; the primers are shown in table S1. All PDI plasmids were transformed into Escherichia coli. Recombinant full-length wild-type human PDI (residues 1-491) and its variant dnPDI were expressed from the vector pET23 in E. coli BL21 (DE3) Codon plus-RIL cells (Novagen, Merck, Darmstadt, Germany). PDI proteins were purified to homogeneity by nickel affinity chromatography as described by Wang et al. (71) . The eluted fractions were dialyzed against 50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl twice to remove EDTA. The PDI proteins were freshly used or stored at -80 °C. SDS-PAGE was used to confirm that the purified human PDI proteins were single species. We used a UV-2550 Probe spectrophotometer (Shimadzu, Kyoto, Japan) to determine the concentrations of wild-type human PDI and dnPDI, using their absorbances at 214 nm with a standard calibration curve drawn by BSA.
A plasmid-encoding, human full-length PDI was a kind gift from Dr. L. W. Ruddock (Faculty of Biochemistry and Molecular Medicine, University of Oulu). The gene for PDI 1-491 was constructed in a prokaryotic expression vector pET23, and a PDI mutant dnPDI was constructed by site-directed mutagenesis using a wild-type PDI template; the primers are shown in table S1. All PDI plasmids were transformed into Escherichia coli. Recombinant full-length wild-type human PDI (residues 1-491) and its variant dnPDI were expressed from the vector pET23 in E. coli BL21 (DE3) Codon plus-RIL cells (Novagen, Merck, Darmstadt, Germany). PDI proteins were purified to homogeneity by nickel affinity chromatography as described by Wang et al. (71) . The eluted fractions were dialyzed against 50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl twice to remove EDTA. The PDI proteins were freshly used or stored at -80 °C. SDS-PAGE was used to confirm that the purified human PDI proteins were single species. We used a UV-2550 Probe spectrophotometer (Shimadzu, Kyoto, Japan) to determine the concentrations of wild-type human PDI and dnPDI, using their absorbances at 214 nm with a standard calibration curve drawn by BSA.
