Fig. 2: Phase separation modulated by vesicle curvature and lipid composition.

a A mixture of recombinant SLP65 with either SUVs (Rh ≈ 20 nm) or LUVs (Rh ≈ 60 nm) was fractionated by a sucrose gradient flotation assay, and the different layers were subjected to anti-SLP65 immunoblot analysis (apparent MW indicated in kDa). Vesicles will float to the top and are found in layers 1 and 2. Bar graph shows the percentage of SLP65 associated with vesicles based on the immunoblot signal intensity. b Confocal fluorescence microscopy of a mixture of different concentrations of Atto 430LS-tagged SLP65_1-330 and CIN85_Δ57 together with SUVs containing DOPC:DOPE of 75:25 mol% (b, top) and DOPC:DOPE:DOPS:cholesterol of 35:25:10:30 mol% (b, bottom), respectively. (Scale bar = 10 μm) Source data are provided as a Source Data file. c SLP65-deficient DT40 B cells were transduced with constructs encoding citrine-tagged SLP65_ΔN fusion proteins with the N-BAR ___domain of Amphiphysin. Wild-type SLP65 and SLP65_ΔN-expressing cells served as positive and negative controls, respectively. The ___domain structures of the expressed SLP65 variants are depicted in the upper part of (c). Cells were loaded with the Ca²⁺-sensitive fluorophore Indo-1 and Ca²⁺ flux was monitored by flow cytometry. Cells were stimulated with anti-BCR antibody (M4) at the indicated time point. d Confocal fluorescence microscopy of cells transduced with N-BAR-SLP65_ΔN were either left untreated (upper panel) or stimulated via their BCR (lower panel).