Figure 6.

Incorporation of distinct ncAAs by film-like organelles enables specific, bioorthogonal protein labeling in cells

(A and B) Schematic overviews of the expected reactions. From a mixture of 3-IF and TCO^∗A-K, only TCO^∗A-K can react with H-Tet-SiR. Analogously, in a separate experiment, from a mixture of SCO-Y and Cbz-K, only SCO-Y can react (B).

(C–F) The doubly tagged imaging reporter (Nup153::EGFP^149TAG::boxB, H2B::mCherry^190TAG::ms2) was expressed together with tRNA^Pyl and one of the indicated optimized GCE systems in presence of indicated ncAAs. The cartoons shown above the images depict the expected results. Top to bottom: EGFP, mCherry, SiR, and merged channel. H-Tet-SiR was used at a concentration of either 200 nM (C–E) or 1 μM (F). The contrast was adjusted for maximum visibility for each condition. Scale bars, 10 μm.