Live-cell imaging of Nrf1/GFP to determine its dynamic movement out of the ER into the cytoplasm.
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Abstract
<p>COS-1 cells were co-transfected with expression constructs for Nrf1/GFP fusion protein and the ER/DsRed marker, and were then subjected to live-cell imaging combined with the <i>in vivo</i> membrane protease protection assay. (<b>A</b>) The cells were permeabilized by digitonin 20 mg/ml) for 10 min, (<b>B</b>) before being co-incubated with PK (50 mg/ml) for 35 min prior to addition of 1% Triton X-100. Over this time interval, real-time images were acquired using the Leica DMI-6000 microscopy system. The merged images of Nrf1/GFP with ER/DsRed are presented (on <i>the third row of panels</i>), whereas changes in the intensity of their signals are shown graphically (<i>bottom</i>). The characteristic features of the arrow-indicated cells are described in the main text. Overall, the images shown herein are a representative of at least three independent experiments undertaken on separate occasions that were each performed in triplicate (nβ=β9).</p