Imaging of fixed and live cells expressing GFP fusion protein with CTD of Nrf1 or its mutants.

Abstract

<p>(<b>A</b>) Schematic of Six expression constructs for the GFP-CTD fusion protein and its mutants; these fusion proteins have been created by attachment of various lengths of CTD of Nrf1 to the C-terminus of GFP. (<b>B</b>) These indicated expression constructs each were transfected into COS-1 cells for 6 h. The cells were then allowed to recover from transfection in fresh medium for 18 h before being fixed by 4% paraformaldehyde and stained for the nuclear DNA by DAPI. The green signals from GFP were observed under confocal microscope and merged with the DNA-staining images. (<b>C</b> and <b>D</b>) Live-cell imaging of GFP-CTD and its mutant GFP-CTD^Δ731–741(lacking its basic c-tail). COS-1 cells had been transfected with expression constructs for either GFP-CTD (<b><i>C</i></b>) or GFP-CTD^Δ731–741 (<b><i>D</i></b>), together with the ER/DsRed marker, before being subjected to real-time live-cell imaging combined with the <i>in vivo</i> membrane protease protection assay. The cells were permeabilized by digitonin 20 µg/ml) for 10 min, before being co-incubated with PK (50 µg/ml) for 30 min. In the time course, real-time images were acquired using the Leica DMI-6000 microscopy system. The merged images of GFP with ER/DsRed are placed (on <i>the third raw of panels</i>), whereas changes in the intensity of their signals are shown graphically (<i>bottom</i>). Overall, the images shown herein are a representative of at least three independent experiments undertaken on separate occasions that were each performed in triplicate (n = 9). The <i>arrow</i> indicates a ‘hernia-like’ vesicle protruded from the cytoplasm.</p