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. 2017 Aug 31;8:1065. doi: 10.3389/fimmu.2017.01065

Figure 5.

Figure 5

Surface plasmon resonance analysis of the effect of Lufaxin on C3b-B and C3b-Bb assembly in the presence of Mg2+ or Ni2+. (A) Lufaxin (MW = 32.5 kDa) injected in combination with factor B or factor B plus factor D stabilizes the formation of proconvertase and convertase complexes on an immobilized C3b surface (3,100 RU). B + Lufaxin = 54 nM factor B + 1 µM Lufaxin, B + D + Lufaxin = 54 nM factor B + 20 nM factor D + 1 µM Lufaxin, B + D = 54 nM factor B + 20 nM factor D, B = 54 nM factor B, and Lufaxin = 1 µM Lufaxin. (B) Lufaxin does not bind to a pre-assembled C3b-Bb complex (C3b surface, 3,000 RU immobilized). Injection mixtures are labeled as in panel (A). “Add Lufaxin” indicates an injection of 1 µM Lufaxin made after assembly of C3b-Bb [B + D]. Trace shows little or no increase in resonance response due to Lufaxin binding. (C) Concentration-dependence of Lufaxin stabilization of C3b-B (C3b surface, 3,500 RU immobilized). Increasing concentrations of Lufaxin were injected over a C3b surface along with 54 nM factor B. Traces in order of increasing amplitude indicate Lufaxin concentrations of 15.6, 31.2, 62.5, 125, 250, 500, and 1,000 nM. (D) Effect of Lufaxin on formation of C3b-B in the presence of Ni2+ (C3b surface, 3,000 RU immobilized). Lufaxin, factor B, and factor B + Lufaxin were injected over a C3b surface in the presence of 2 mM NiCl2. The plots are labeled as in panel (A). (E) Binding of Lufaxin to a preformed C3b-B complex in the presence of Ni2+ (C3b surface, 3,000 RU immobilized). The C3b-B complex was formed on the same surface as in panel (D) (line B), and during the dissociation phase, 1 µM Lufaxin was injected. (F) Comparison of C3b-B stabilization by Lufaxin in the presence of Mg2+ and Ni2+. Factor B + Lufaxin [as in panel (A)] was injected over the same C3b surface using HBS-N containing MgCl2 or NiCl2 as the sample and running buffer. (G) Stabilization of C3b-B by Lufaxin in the absence of divalent cations (C3b surface, 3,150 RU immobilized). Injections were made in HBS-N buffer alone. Traces are labeled as in panel (A).