Figure 1
From: Establishment of a system for monitoring endoplasmic reticulum redox state in mammalian cells
Characterization of MERO-GFP (Mammalian endoplasmic reticulum-localized redox-sensitive green-fluorescent protein). (a) Schematic structure of MERO-GFP. (b) Confocal imaging of MERO-GFP in COS7 cells. ER was visualized using DsRed-ER tracker. (c) Excitation spectra of MERO-GFP treated with different concentrations of dithiothreitol (DTT). (d) Emission spectrum of oxidized MERO-GFP in untreated NSC34 cells. The wavelength of excitation was 394 nm. (e) Emission spectrum of reduced MERO-GFP in NSC34 cells treated with 2 mM DTT. The wavelength for excitation was 473 nm. (f) Real-time imaging of MERO-GFP signal from 476 nm excitation and 405 nm excitation in INS-1 832/13 cells treated with or without 2 mM DTT. 476/405 ratio images are displayed in false colors in the lookup table. (g) MERO-GFP ratio in cells treated with the indicated concentrations of DTT and H2O2. MERO-GFP ratio was determined by a plate reader. Data represent means±s.d. from three independent experiments. (h) Real-time monitoring of MERO-GFP ratio in live NSC34 cells treated with DTT. NSC34 cells expressing MERO-GFP were treated with DTT (1 mM), and the fluorescence from MERO-GFP was measured every minute. Ten minutes after the treatment, DTT was washed out. The red line indicates the fluorescence intensity with excitation at 394 nm. The blue line indicates the fluorescence intensity with excitation at 473 nm. Black line indicates the normalized MERO-GFP ratio. (i) AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid)-modified SDS-PAGE of MERO-GFP. All error bars shown, ±s.d.
