Fig. 2. Engineering of enhanced miRFPs.

a Alignment of N-termini of monomeric NIR FPs derived from RpBphP1 and RpBphP2. b Effective brightness of miRFP703 variants with 12–16 amino acid residues removed from the N-terminus. Analysis was performed in COS-1 cells using flow cytometry 72 h after transfection. The effective brightness of the original miRFP670 was assumed to be 100% for normalization. c Effective brightness of miRFP670 and miRFP703 and their variants with 13 amino acid residues removed from N-terminus in control cells and after 4 h incubation with 10 µM proteasome inhibitor bortezomib or 50 µg ml⁻¹ ribosome blocker cycloheximide in live COS-1 cells. Error bars, double s.e.m. (n ≥ 3; transfection experiments). Brightness of FPs in control cells was assumed to be 100% for normalization. d Effective brightness of miRFP670 and miRFP703, their variants with 13 amino acid residues removed from N-terminus, and their variants with N-termini of RpBphP2. Measurements were performed in COS-1 cells 72 h after transfection. Effective brightness of miRFP720 was assumed to be 100% for normalization.