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Synblock solution (BUF034) used for blocking non-specific binding sites in a Mincle binding assay
T. forsythia S-layer binds Mincle.
(A) Binding of S-layer to Mincle. T. forsythia S-layer immobilized in microtiter plate wells was incubated with Mincle-Fc fusion protein or MMR-Fc in lectin binding buffer (TBS, 5 mM CaCl2). As control, ManLAM was immobilized in wells. After washing wells with buffer, binding was detected with HRP-coupled goat anti-human IgG-Fc and chromogenic substrate TMB. Color was read by measuring absorbance at 450 nm. As shown, S-layer bound Mincle in a dose dependent manner and MMR as control bound ManLAM but not S-layer. (B) Mincle binding to T. forsythia S-layer is Ca2+-dependent. To analyze the Ca2+ dependency of Mincle binding to S-layer, Mincle-Fc was incubated with plate bound S-layer in Ca2+ (TBS, 5 mM CaCl2) or EDTA containing buffer (TBS, 10 mM EDTA). Plate bound TDB was used a control ligand or Mincle binding. Binding was then detected as above. (C) S-layer dose dependently inhibited binding of Mincle to TDB. Plate bound TDB was incubated with Mincle-Fc in lectin binding buffer with increasing concentrations of S-layer. Wells were washed with elution buffer and binding was measured as above. Each value represents the mean (± SD) of 3 values measured in one representative assay. Data are representative of three independent experiments with similar results; *, P<0.05.
From: Chinthamani S, Settem RP, Honma K, Kay JG, Sharma A (2017)
Macrophage inducible C-type lectin (Mincle) recognizes glycosylated surface (S)-layer of the periodontal pathogen Tannerella forsythia.
PLoS ONE 12(3): e0173394.