Western blots of gel-shift assay with sfGFP-N150tag/Tet3-Butyl and sTCO-PEG5K. (A) Extracts of sfGFP-N150tag/Tet3-Butyl expressed in HEK293T/17 pulse-labeled with sTCO-PEG5K. Lane 1 represents sfGFP-150tag expressed in the presence of NES-R284 (pAcBac1) and Tet3-Butyl ncAA. Lane 2 demonstrates that the addition of sTCO-PEG5k for 3 min induces a gel shift in the molecular weight band, and lane 3 shows the molecular weight shift of the sfGFP-N150tag after 10 min of incubation with sTCO-PEG5K. (B) Extraction of sfGFP-N150tag-FLAG/Tet3-Butyl with RIPA buffer and subsequent with (+) and without (−) sTCO-PEG5K labeling for 10 min before quenching with 1 mM Tet2 is shown after blotting for the C-terminal FLAG tag. (C) Control experiment of TRPV1-T468tag-GFP with pAcBac1 alone or supplemented with Tet3-Butyl in culture media. Ca²⁺ imaging of TRPV1 activity done as described in methods using Fluo4-AM and 500 nM capsaicin. All images are normalized to the brightness of ionomycin image. A single responding cell in the absence of Tet3-Butyl (−) was qualitatively deemed an outlier since cells otherwise showed no activity compared with Tet3-Butyl–treated cells (+), which showed robust activity with capsaicin. Source data are available for this figure: SourceData FS4.