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. 2020 Dec 8;11:596103. doi: 10.3389/fimmu.2020.596103

Figure 3.

Figure 3

LDLR binds to both opsonized and unopsonized crystals. (A) MSU crystals (lot1-3) were opsonized in different solutions (unopsonized (HBSS), human serum, murine serum, or HBSS containing 5% BSA, 1 mg/ml fibrinogen, or 1 mg/ml LDL) at 37°C for 30 min. After washing with HBSS, the particles were incubated with 5 µg/ml recombinant His-tagged protein in HBSS + 5% BSA at 4°C for 60 min (hLDLR, mLDLR, hMARCO, hCD32b). Bound proteins were stained with anti-His Tag PE and analyzed using a flow cytometer. An unpaired, two-sided t-test was used for statistical evaluation (ns = not significant, *p < 0.05, **p < 0.01 ); MFI and SEM are shown. (B) Calcium carbonate and m-CPPD crystals were pre-incubated in HBSS or HBSS-containing 1 mg/ml hLDL and then incubated with hLDLR and stained as described in (A). An unpaired, two-sided t-test was used for statistical evaluation (ns = not significant, **p < 0.01). Mean MFI and SEM of n=3 samples is shown. (C) Indicated crystals were opsonized in human serum at 37°C for 30 min. Incubation with recombinant LDLR was done as in (A). Protein binding was analyzed using mouse anti-His Tag plus goat anti-mouse AlexaFluor488. Fluorescence of the samples was detected by fluorescent microscopy; scale bar = 40 µm. Representative of three independent experiments. (D) Indicated crystals were opsonized with human serum at 37°C for 30 min. After incubation, particles and supernatant were separated by centrifugation: the supernatant was collected, while the particles were extensively washed with HBSS to remove unbound proteins. Bound proteins were eluted. Both supernatants and eluates were subjected to Western blot analysis using ApoB, ApoAI, and ApoE antibodies. (E) Two distinct preparations of MSU crystals (lot1, lot2) were incubated with human serum or LDL-depleted human serum (both containing 10 µg/ml CRP) at 37°C for 30 min. Incubation with recombinant proteins (hLDLR and hCD32b) as well as detection and analysis of the bound proteins was performed as described in (A); MFI and SEM are shown. CRP binding was analyzed using CRP antibody and anti-rabbit-AlexaFluor488. Fluorescence of the crystals was detected by flow cytometry (MFI anti-CRP +SEM). A paired, two-sided t-test was used for statistical evaluation. (*p < 0.05, **p < 0.01, ***p < 0.001).