Fig. 6
From: A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo
Tracking single mRNA translation in living U2OS cells. a A diagram depicting frankenbody (FB-GFP; green) and MCP-HaloTag-JF646 (magenta) labeling HA epitopes and mRNA stem loops, respectively, in a KDM5B translation reporter. b A representative cell (10 cells in three independent experiments) showing colocalization of FB-GFP (green) with KDM5B mRNA (magenta). See also Supplementary Movie 3. c A representative cell (upper-left, 9 cells in three independent experiments) showing the disappearance of nascent chain spots labeled by FB-GFP within seconds of adding the translational inhibitor puromycin. See also Supplementary Movie 4. Upper-right: The mean number of nascent chain spots normalized to pre-puromycin levels decreases while mRNA levels remain constant (9 cells from three independent experiments). Error bars, cell-to-cell SEM. Lower: a sample single mRNA montage. d A diagram depicting FB-Halo-JF646, FB-mCh, or FB-SNAP-JF646 labeling HA epitopes in a KDM5B translation reporter. e, f Representative cells (three cells in two independent experiments for both FB-Halo and FB-mCh), single mRNA montages, and quantification, as in c, showing the loss of nascent chain spots labeled by e FB-Halo-JF646 or f FB-mCh upon puromycin treatment. See also Supplementary Movies 5 and 6. Scale bars, 10 µm. Source data are provided as a Source Data file
