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Figure
1. ReAsH labeling of the TC motif in the absence of cycloheximide results in a pattern similar to GFP–ß-actin. (A) Schematic representation of the time course of the procedure used in B and C. (B) Epifluorescence image of the distribution of GFP–ß-actin from a C2C12 cell transfected with the FL construct. (C) Epifluorescence image of ReAsH staining from the cell in B. Note the similarity between the staining patterns in the absence of cycloheximide (compare B and C). (D) Model of the translation site detection scheme. ReAsH (red) will label the TC motif of nascent chains of ß-actin and mature ß-actin, whereas GFP will only label mature ß-actin protein. ReAsH staining of cells containing the TC–GFP–ß-actin constructs performed in the presence of cycloheximide should increase the number of nascent chains at the polysome site as the rate of ribosomal translocation decreases, providing a more robust and persistent fluorescence signal, resulting in bright red puncta at the sites of translation. Ribosome packing is not to scale. Bar, 10 µm.
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