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. 2021 Nov 20;120(24):5454–5465. doi: 10.1016/j.bpj.2021.11.2884

Figure 1.

Figure 1

Representative transmembrane protein is immobilized in the supported bilayer but fully mobile in the GUV membrane. (A) Confocal fluorescence images of the lipids in the same GUV membrane or membrane-coated bead (MCB) before and after NBD bleaching by dithionite treatment (left), with their normalized average fluorescence intensities shown in the right panel. The error bar indicates the standard deviation. (B) Confocal fluorescence images of the same GUV or MCB taken before (t = 0) and after photobleaching at the indicated time. (C) Fluorescence intensities as a function of time after photobleaching (symbols) and their best fits (dashed curves) to determine the diffusion coefficients of lipids or VAMP2 as indicated. The intensities were normalized by the corresponding intensities just before photobleaching. (D) Fluorescence images of Alexa Fluor 647-labeled VAMP2 in the GUV or MCB taken before (t = 0) and after photobleaching. The GUV or MCB membranes used in the FRAP experiments contained 99.65 mol % POPC, 0.25 mol % NBD-DOPS, and 0.1 mol % Alexa Fluor 647-labeled VAMP2. All GUVs encapsulated 30% (w/v) iodixanol.