Supplementary Figure 2: CD28 cytoplasmic domain specifically binds to acidic phospholipids.
From: Dynamic regulation of CD28 conformation and signaling by charged lipids and ions
(a) Alignment of CD28CD from different species. Most residues are highly conserved, especially the two important YxxM and PYAP signaling motifs (marked in red) and basic residues (marked in green). The residue conservation is visualized by the sequence logo of CD28CD generated from the Skylign web server http://www.skylign.org.
(b) Net charges of human and mouse CD28 cytoplasmic domains at pH 7.0.
(c) The protein sequences of mouse CD28CD and three mutants used in (d-g). Two polybasic regions (PBR) are marked in red, and mutant sites are marked in blue.
(d-g) Binding of CD28CD WT and mutants to acidic phospholipids was measured by the equilibrium-based microdialysis assay.
(d) A cartoon illustration of the equilibrium-based microdialysis assay. See Online Methods for details.
(e) Binding of CD28CD WT to different acidic phospholipids measured by the microdialysis assay. LUVs in the experimental chamber were composed of either 100% of the acidic phospholipids POPG, POPS, PI, PA, or a lipid mixture (40% POPC, 20% POPS, 20% POPE, 10% POPG and 10% PI). LUVs in the control chamber were composed of 100% of the zwitterionic lipid POPC. CD28CD-Alexa 488 was used at a concentration of 10 nM and LUVs at 5 mM. Two to three independent samples were measured for each condition and the results are plotted as the percentage of the fluorescence intensity in the experimental and control chambers.
(f-g) Binding of CD28CD WT and mutants to acidic phospholipids measured by the equilibrium-based microdialysis assay. Two to three independent samples were measured for each condition and the results are plotted as the percentage of the fluorescence intensity in the experimental and control chambers (F), and are further converted to the binding efficiencies of the mutants to POPG (G). Data were analyzed by unpaired t-test. **P < 0.01; ***P < 0.001; ns: no significant difference.
Data are representative of three (e) or seven (f, g) independent experiments. The center value and error bar in e-g denote mean and range. Source data for e-g are available in Source Data 4.
