Specificity experiments with 2G4C. (A) Z-projection of confocal image stacks of U2OS cells stably expressing A1aY1 binder tagged with TagRFP-T at the N terminus in magenta and 2G4C tagged with EGFP at the C terminus in gray. The cells were additionally stained with SiR-tubulin (green). Line scan for the yellow line shows the intensity profile of 2G4C-EGFP, TagRFP-T_A1aY1, and SiR-tubulin fluorescence. (B) Confocal stacks of the cells expressing 2G4C alone, with vasohibin2-X1-GFP-2A-SVBP and TTL5 showing retention of binding of 2G4C. The line scan represents microtubule signal from the region of the cell marked in yellow. Scale bars = 10 µm. A.U., arbitrary units; Fl., fluorescence.