ISLAD colocalizes with T-cell membranes with high membrane binding affinity. T cells were loaded with the membrane fluorescent dye DiD or with the cytoplasmic fluorescent dye CMTMR, and then fluorescent NBD-ISLAD was added to cells. Cellular localization of the peptides in mMOG35-55 T cells (A) and in Jurkat T cells (B) were observed using confocal microscopy. The left column is the fluorescence of NBD-ISLAD alone (green). The middle column is the fluorescence of the cytoplasmic or membrane dye (red). The right column is the merged image of them. (C) The graphs shows the percentages of colocalization ± SD between ISLAD and cell membrane (black) or between ISLAD and cytoplasm of the cell (gray) in T cells. (D) The graph shows the fluorescent measurements of ISLAD-membrane interactions. Peptides were titrated with increasing concentrations of PC/Chol (9:1) LUVs, and changes in fluorescence anisotropy of their intrinsic Trp were measured. The fitting curve from the nonlinear least-squares model is presented (supplemental methods, Equation 1), which gives the membrane binding affinity constant. Scale bars represent 2 µM. **P < .01. a.u, arbitrary units.