Fig. 4. Lysate fluorescence analysis of Acd incorporation in mammalian cells. Top: fluorescence measured from EGFP-Y₄₀δ HEK cell lysates after 36 h using Mb AcdRS 41 or 82 with 300 μM Acd or no ncAA added. As a negative control, the EGFP plasmid was transfected without the AcdRS and tRNA_CUA plasmid (−aaRS). WT EGFP was separately transfected as a positive control. In all conditions, DN-eRF1 was also transfected. n = 3. Protocols are described in detail the ESI† comparison of AcdRS incorporation efficiency section. Cell images are shown in ESI, Fig. S21.† Bottom: expression of EGFP-Y₄₀δ using Mb AcdRS 82 was performed as above, with varying concentrations of Acd. n = 3. Protocols are described in the ESI† optimizing expression conditions section. Cell images are shown in ESI, Fig. S3.†.