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. 2014 Sep 25;289(45):31188–31199. doi: 10.1074/jbc.M114.602961

FIGURE 2.

FIGURE 2.

Dynamic regulation of Ero1α by redox active PDI in cells. A, HeLa transfectants expressing Ero1α C99/104A-myc were co-transfected with PDI WT (+) or empty (−) plasmid. Left panel, the expression level of PDI was visualized by αPDI WB. Right panel, HeLa cells were exposed to 150 μm DTT, aliquots were taken at indicated times and analyzed by nonreducing SDS-9% PAGE and αmyc WB. B, to maximally block the active sites of PDI, HeLa transfectants expressing Ero1α C99/104A-HA were treated by 50 μm DTT and 50 μm 16F16 dissolved in DMSO for 8 h before harvest. Parallel treatments by 50 μm DTT alone (control) or combined with DMSO were also carried out. The shift of part of Ero1α to Ox1 at steady state was due to the pre-treatment by 50 μm DTT. The activation of Ero1α was monitored as in A and visualized by αHA. C, HeLa cells were treated with control (−) or shPDI (+) plasmid and then co-transfected with Ero1α C99/104A-myc. Left panel, the knockdown efficiency of PDI is shown by αPDI WB. Right panel, after a 10 mm DTT pulse treatment, cells were washed to remove DTT and followed by a chase at indicated times. Cell lysates were analyzed by nonreducing SDS-9% PAGE and αmyc WB. D, HeLa transfectants expressing Ero1α C99/104A-HA were treated as in B, and the inactivation of Ero1α was monitored as in C and visualized by αHA. E, left panel, expression level of endogenous Ero1α in HeLa cells transfected with control (−) or shEro1α (+) plasmid was monitored by WB using αEro1α. Right panel, HeLa cells were treated with control (−) or shEro1α (+) plasmid and then co-transfected with Ero1α C99/104A-HA, which has an altered codon bias to the endogenous ERO1 gene and is not targeted by the shEro1α. The redox states of Ero1α C99/104A-HA during the pulse-chase process were monitored as in C and visualized by αHA. F, HeLa transfectants expressing Ero1α C99/104A-HA were co-transfected with the hyperactive Ero1α C104/131A-myc (+) or empty (−) plasmid. Left panel, the expression of Ero1α C104/131A-myc was visualized by αmyc WB. Right panel, the redox states of Ero1α C99/104A-HA during the pulse-chase process were monitored as in C and visualized by αHA. The Red, Ox1, and Ox2 species of Ero1α were indicated on the right margin of each panel.