Figure 1.

Optical and physiological sensitivity of roGFPiE to the ER redox environment. (A) Excitation (measured by the emission at 520 nm) and emission (measured by excitation at 450 nm) scans of purified roGFPiE dithiol (SH) or disulfide (S-S; 1 µM) performed at a spectral resolution of 1 nm. (B) Time-dependent changes in the ratio of fluorescence emission (excitation: 470 nm vs. 395 nm) of the fully reduced purified roGFPiE dithiol (1 µM) introduced at t = 0 into a glutathione redox buffer (5:1 GSH/GSSG, 4 mM total) in the absence or presence of 30 µM PDI. The emission ratio of roGFPiE maintained continuously in the presence of the reducing agent DTT (5 mM) or the oxidizing agent lipoic acid (5 mM) is provided as a reference for the dithiol and disulfide forms, respectively. (C) Initial velocity of the oxidation of roGFPiE from the dithiol to the disulfide in the presence of the indicated concentration of PDI, calculated from the linear phases of measurements as in B. (D) Fluorescent photomicrographs of COS7 cells expressing ERroGFPiE, stained with rabbit polyclonal anti-GFP, mouse monoclonal anti-PDI, and chick polyclonal anti-BiP (as ER markers). The nucleus was visualized by Hoechst staining (in blue in the merged right-most panel). (E) Immunoblot of ERroGFPiE expressed in untreated or DTT-treated (5 mM DTT for 10 min) HEK 293T cells, resolved by nonreducing (NR) and reducing (R) SDS-PAGE. Shown are representative experiments reproduced three times (A–D) or twice (E).