Figure 2.

In vitro binding characteristics of aptamer AptMincle in comparison with antibody

(A) Immunofluorescence imaging of the relative expression of pSyk^Y525 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 (Mincle^low) and LPS-primed (Mincle^high) J774.1 macrophages co-stained with anti-CLEC4E antibody (InvivoGen, USA) and 5′-Cy3-conjugated AptMincle. (C) Dose-dependent inhibition of Syk^Y525 phosphorylation in J774.1 macrophages by anti-Mincle antibody (InvivoGen) compared with AptMincle. (D) Whole-cell ELISA of (i) pSyk^Y525/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 AptMincle^DRBL 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.