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. 2020 Jun 9;31(10):107697. doi: 10.1016/j.celrep.2020.107697

Figure 2.

Figure 2

NCX1 Localization to Lipid Microdomains

(A) Visualization of cellular cholesterol with filipin in control (left), SDS-treated (center), and MβCD-treated (right) HEK293 cells (scale bar, 20 μm).

(B) Representative FRET images in HEK293 cells expressing WT (upper) and unpalmitoylatable (C739A, lower) NCX1 FRET sensors following treatment to enhance (SDS) or reduce (MβCD) raft formation (scale bar, 10 μm).

(C) Mean FRET data. Enhancing raft formation increases NCX1-NCX1 FRET and reducing raft formation decreases NCX1-NCX1 FRET, but only when NCX1 is palmitoylatable. ∗∗∗∗p < 0.0001, calculated by unpaired t test. N = 14 (WT), 35 (WT+SDS), 16 (WT+MβCD), 19 (C739A), 26 (C739A+SDS), and 14 (C739A+MβCD).

(D) Schematic representation of giant plasma membrane vesicle (GPMV) phase separation and representative images displaying phase-separation behavior in the presence of WT NCX1 (upper) and C739A NCX1 (lower). Scale bar, 2 μm.

(E) Colocalization analysis in phase-separated GPMVs. Left: increased colocalization of raft (CTxB-Alexa Fluor 647) and non-raft (FAST-DIL) markers in the presence of WT versus C739A NCX1. Center: raft colocalization of WT and unpalmitoylatable NCX1. Right: non-raft colocalizaton of WT and unpalmitoylatable NCX1. ∗∗∗∗p < 0.0001, calculated by unpaired t test. N = 27 (WT) and 28 (C739A).