TRPV1-tag construct expression with GCE4All-Tetv3.0 system. (A) Western blot of TRPV1-K464tag-GFP and TRPV1-T468tag-GFP with pAcBac1 and Tet3-Butyl ncAA. Addition of sTCO-PEG5k (PEG5k) used to test access to tetrazine ncAA in TRPV1-tag target. Excess Tet3-Butyl (Tet-bu) is added to quench sTCO-PEG5k at the 10 min point to prevent off-target labeling with PEG5K. TRPV1-tag-GFP molecular weight is estimated as ∼120 kD (see yellow asterisk) and TRPV1-tag-GFP/sTCO-PEG5K would be upshifted to ∼125 kD (see red arrow). (B) Ca²⁺ imaging experiment to test the functionality of TRPV1-T468tag-GFP expressed with GCE4All-Tetv3.0. Median value of the set is indicated with a purple triangle. Significant difference was observed between the Ca²⁺ response evoked by 500 nM capsaicin in TRPV1-T468tag-GFP cells (green) and non-responsive cells (black) in the same imaging experiment (Wilcoxon rank sum; P < 0.001). Only results of Ca²⁺ imaging with the T468tag construct are shown because labeling experiments with this construct and sTCO-Cy5 are demonstrated in panels C and D. (C) HEK293T/17 cells expressing TRPV1-T468tag/Tet3-Bu-GFP (cyan) are labeled with sTCO-Cy5 (magenta). (D) Correlation of GFP brightness in the image of TRPV1-T468tag/Tet3-Bu-GFP expressing cells that are labeled with 500 nM sTCO-Cy5 for 5 min as described in methods. Five cells expressing various levels of the TRPV1-T468tag/Tet3-Bu-GFP construct as determined by GFP intensity show correlated labeling with sTCO-Cy5 (linear fit, r² = 0.96; P value = 0.0034; see Fig. S5 and Materials and methods for description of statistics). Source data are available for this figure: SourceData F3.