Extended Data Fig. 5: ApoE binds to the activated C1q; LDLR and C1sC1r tetramers do not compete with C1q-ApoE binding.
From: ApoE attenuates unresolvable inflammation by complex formation with activated C1q
a, ApoE-C1q interaction is dependent on Ca2+. Real-time binding of ApoE to C1q was followed using biosensor analyses. Binding of ApoE to C1q is reduced in a dose-dependent manner upon increasing amounts of EGTA (0.1–3 mM). b,c, co-immunoprecipitation of C1q-ApoE complexes; (b) anti C1q antiserum precipitate C1q-ApoE complexes composed of purified proteins with activated C1q, but not with inactive C1q from NHS; (c) Anti-ApoE antiserum precipitates C1q-ApoE complexes but no complexes from NHS. C1q-ApoE complexes were eluted with glycine buffer, then, C1q or ApoE proteins were separated by SDS-PAGE and immunoblotted using goat anti-C1q antiserum (left panel of b, and c) or goat anti ApoE antiserum (right panel of b) separately. Full scanned blot images in b,c are available from source data figures. d, ApoE peptide P139-152 but not ApoE peptide P30-40 competes with immobilized ApoE3 for binding to C1q in a dose-dependent manner. e, C1q antibody binding to C1q is not affected by SDS. f, C1q and LDLR bind simultaneously to ApoE. 20 nM C1q was incubated with increasing concentrations of LDLR to immobilized ApoE and binding of C1q and LDLR was followed by ELISA. Background binding of anti C1q and anti LDLR antisera to immobilized ApoE were set as 0%. g, ApoE does not compete with C1sC1r tetramers for binding to C1q. C1q in addition to increasing amounts of C1sC1r tetramers was added to immobilized ApoE3 and C1q binding was determined. Data in d-g represent means ± s.e.m. of at least three independent experiments. Two-tailed Student´s t test. Data in a,b,c are representatives of 3 independent experiments.
