Extended Figure 4. ApoE binds to C1q but not to other complement components.

(a) ApoE isoforms bind to the C1 complex, but not to C4 or C2. Biotinylated ApoE was immobilized on streptavidin-coated sensors and incubated with C1 complex, C4, C2, or buffer; (b) The C1 complex binds to immobilized ApoE isoforms. (c) ApoE isoforms bind to C1 and factor H, but not to C3 or C3b; (d) Normal human serum (NHS)-derived C1 binds to immobilized plasma-purified ApoE3 and to recombinant ApoE isoforms; (e) C1q binds to immobilized plasma-purified ApoE3 and to all recombinant ApoE isoforms; (f) Plasma-purified C1q was coated on a sensor chip (CM5) and plasma-derived ApoE (62-1000 nM) was injected into the fluid phase (75 mM NaCl, 5 mM HEPES, 1 mM CaCl2). (g) Mannose-binding lectin (MBL) does not bind to C1q as determined by biolayer interferometry; (h) Apolipoprotein A (ApoA) does not bind to C1q as determined by biolayer interferometry. (i) C1q-ApoE complexes revealed by proximity ligation assay (PLA) on cultured human apoptotic cells (THP-1) were detectable when treated with NHS but not with C1q-depleted serum (dNHS). Data represent mean fluorescence intensity (MFI) ± SEM of 16 cells for each group. Bar 10 µm. Data in b,c,d,e represent means ± SEM of at least three independent experiments. Data a,f,g and h represent means of at least two independent experiments. Two-tailed Student's t-test.