Fig. 4.

Redox imaging demonstrated that the cytoplasmic surfaces the plasma membrane and vesicular membrane are characterized by different levels of glutathione oxidation and H₂O₂ production. A, Grx1-roGFP2^Palm was transfected into HeLa cells and I405/I488 time-lapse imaging was performed by live-cell confocal microscopy. R value (I405/I488) represents the degree of sensor oxidation and shown in a false-color scale. Time is indicated in each image (h:m:s). Scale bar, 10 µm. Signals from the plasma membrane (PM; open arrow heads) and vesicular membranes (VM; filled arrow heads) are indicated. Time-lapse movie is available as Supplementary Data 5. B and D, Grx1-roGFP2^Palm and roGFP2-Orp1^Palm were transfected into HeLa cells and steady-state I405/I488 images were taken (z-sections). X-Y image in the right corresponds to the focal plane indicated by *. Signals from the plasma membrane (PM; open arrow heads) and vesicular membranes (VM; filled arrow heads) are indicated. C and E, Average R_I405/I488 values from multiple cells (n = 5). PM, plasma membrane. VM, vesicular membrane.