Fig. 8

The GCE-tag is versatile and in some cases can be further minimized. a Western blot analysis of HEK293T cells transfected with pBUD-Pyl-RS plasmid inserted with GFP-SKL N-terminally tagged with (from left to right) the optimized GCE-tag (HA-GGSG-ncAA), tags with alternative short epitopes FLAG-GGSG-ncAA and Myc-GGSG-ncAA, and a tag with no epitope (GGSG-ncAA). Cells were incubated for 48 h in the presence of BCN-Lys, harvested and subjected to western blot analysis using anti-GFP antibodies. b–d Maximum intensity projections taken from live-cell images of COS7 cells transfected with FLAG-GGSG-ncAA (upper panels) or GGSG-ncAA (lower panels) conjugated to GFP-SKL (b), Exo70 (c), or α-tubulin (d) and labeled with the indicated tetrazine dyes. Plots in b represent co-localization analysis between intensity values of the 640 (SiR) and 488 (GFP) channels performed on large subsets of the cells that exclude the nucleus (correspond to rectangles in merged image); Pearson’s correlation values: upper panel, 0.87; bottom panel, 0.76. c, d Left, maximum intensity projection of representative cells. Right, line intensity profiles measured for the dashed lines in left panels. Results presented in each panel were obtained in at least 3 independent experiments. Scale bar 10 μm