Emission spectrum of ACP-GPI. (A) Our system recognizes a difference in the local membrane environment between ACP-GPI produced in WT and PGAP2/3 mutant cells. Cells transiently expressing ACP-GPI in WT or PGAP2/3 mutant cells were stained with CoA-PEG11-NR and the emission spectrum between 561 and 695 nm was measured from six images (n = 35 for WT and n = 36 for PGAP2/3 mutant). (B) The emission spectrum of the bulk plasma membrane assessed by NR12S was identical between WT ACP GPI expressing cells and PGAP2/3 mutant ACP-GPI expressing cells. The data represents mean ± SEM from six images (n = 36 ROIs for each sample).