C1q, HMGB1, RAGE, and LAIR-1 form a multimolecular complex in lipid rafts. (A) SPR assay of C1q and RAGE binding; K_D = 855 nM. Experiments were repeated 3 times. (B) SPR assay of RAGE-C1q-HMGB1 trimolecular complex. RAGE (500 nM) was immobilized onto a CM5 chip and the first analyte (C1q, 200 nM) was added to saturation. HMGB1 (500 nM) was added to the RAGE-C1q complex in multiple pulses (left). Alternatively, HMGB1 was added to immobilized RAGE until the chip was saturated, followed by C1q in multiple pulses (right). N = 3. (C) SPR assay for HMGB1 and C1q binding; K_D = 200 nM. N = 4. (D) SPR assay of HMGB1 in different redox states and C1q binding. C1q (200 nM) was immobilized onto a CM5 chip, and disulfide-, all thiol-, or oxidized-HMGB1 (500 nM) was added. N = 3. (E) C1q-coated beads were incubated with saturating amounts of HMGB1, followed by 250 ng or 500 ng of RAGE. Complexes were analyzed by western blot using anti-RAGE, anti-Cbp tag antibody for HMGB1 or infrared-labeled streptavidin for biotinylated-C1q. N = 3. ***P < .001 (one-way ANOVA). (F) Human monocytes were treated with HMGB1 (3 μg/mL) in the absence or presence of C1q (25 μg/mL) for 15 minutes at 37°C. Colocalization of LAIR-1 and RAGE on the plasma membrane was assessed by PLA. Red dots (PLA positive), representing colocalization of RAGE and LAIR-1, are only seen in the presence of C1q, with or without HMGB1. Percentage of PLA-positive cells in total cells was counted in several random fields in 3 independent experiments. (G) PLA assay using C1 (25 μg/mL) complex instead of C1q. N = 3. (H) Lipid raft fractions from monocytes treated with HMGB1 (3 μg/mL), in the absence or presence of C1q (25 μg/mL), for 15 minutes at 37°C were concentrated and analyzed by western blot for LAIR-1, RAGE, HMGB1, C1q, or Flotillin1 as a lipid raft marker. N = 3. DAPI, 4′,6-diamidino-2-phenylindole.