Effect of emission spectrum of CoA-PEG-NR bound ACP-IR to PEG-linker length and sensitivity test to membrane-voltage change. (A and B) Sensitivity of emission spectrum to PEG-linker length. Cells expressing 2031-ACP-IR (A) or 1992-ACP-IR (B) were labeled by CoA-PEGn-NR (n = 5, 11, 14, or 27) or NR12S and emission spectrum was analyzed (each sample: n = 75 from five images). (C) Membrane depolarization led to decreased fluorescent intensity of CoA-PEG11-NR bound 2031-ACP-IR.2031-ACP-IR (n = 9) or 1992-ACP-IR (n = 9) expressing cells labeled by CoA-PEG11-NR and the change in fluorescent intensity induced by membrane depolarization (100 mV) using patch-clump was recorded. Data are means ± SEM. Statistical significance was calculated by a two-sided t test, the change was significant ****, P < 0.0001. (D and E) Correlation between the membrane localization of dye and voltage sensitivity. (D) Cells expressing 1992-ACP-IR were stained with CoA-PEG5-, 11-, or 27-NR and the fluorescent intensity was measured upon membrane depolarization. Data represent means ± SEM. of n = 5 (PEG5), 9 (PEG11), and 18 (PEG27). Statistical significance was calculated by one-way ANOVA; ns, not significant and significant *, P < 0.05. (E) Cells expressing 2031-ACP-IR were stained with CoA-PEG5-, 11-, or 27-NR and the fluorescent intensity was measured upon membrane depolarization. Data represent means ± SEM of n = 12 (PEG5), 9 (PEG11), and 11 (PEG27). Statistical significance; ns.
