Fig 1: In vitro binding characteristics of aptamer AptMincle in comparison with antibody(A) Immunofluorescence imaging of the relative expression of pSykY525 and Mincle in unstimulated, TDB-stimulated (50 μM), LPS-primed (10 ng/mL, 24 h), or LPS → TDB (10 ng/mL LPS + 50 μM)-stimulated J774.1 macrophages (left to right). (B) Staining of a heterogeneous population of control (Minclelow) and LPS-primed (Minclehigh) J774.1 macrophages co-stained with anti-CLEC4E antibody (InvivoGen, USA) and 5′-Cy3-conjugated AptMincle. (C) Dose-dependent inhibition of SykY525 phosphorylation in J774.1 macrophages by anti-Mincle antibody (InvivoGen) compared with AptMincle. (D) Whole-cell ELISA of (i) pSykY525/Syk and (ii) pP65/P65 relative expression in LPS-primed, TDB-treated J774.1 macrophages when challenged with AptMincle (1 μM). (E) Whole-blood assessment of AptMincleDRBL presence in mice via fully quantitative RT-PCR. IC50 was calculated on GraphPad Prism 8 using a non-linear fit: y = 100/(1 + x/IC50). One-way ANOVA with Tukey’s post hoc test was performed for multiple non-parametric data comparisons. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Scale bar = 10μm.
Fig 2: Characterization of the in vitro function of aptamers with Mincle affinity(A) Binding characteristics of AptMincle, and its modified counterparts, with rhMincle as determined by ELONA. (B) Graphical depiction of the modifications made to the aptamer sequences (black, original; white, modified). (C) The predicted secondary and tertiary structures and docking simulation of AptMincleCORE (purple) with Mincle (white). Box: highlighted predicted region of interaction surrounding a calcium molecule. (D) The predicted secondary and tertiary structures and docking simulation of AptMincleRND (purple) with Mincle (white). Box: highlighted predicted region of interaction surrounding a calcium molecule. Dissociation constants were calculated on GraphPad Prism 8 using a non-linear regression binding analysis with assumed one-site target parameters.
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