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. 2021 Feb 18;4:229. doi: 10.1038/s42003-021-01745-7

Fig. 4. CLEC18A and CLEC18A(S339R) associate with TLR3 and bind poly (I:C) directly.

Fig. 4

a, b Human macrophages (2 × 105) were incubated with HMW poly (I:C) or H5N1 virus (MOI = 2) for 2 h, followed by incubation with mouse anti-human CLEC18 mAb and rabbit anti-TLR3 mAb. After washing, samples were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG mAb (donor) and Alexa Fluor 546-conjugated goat anti-rabbit IgG mAb (acceptor). Cells were examined under a confocal microscope (TCS-SP5-MP-SMD, Leica), and images were analyzed by the “SymPhoTime” software. The image of the fluorescence lifetime is shown by false colors. Green color represents a long lifetime of CLEC18A, whereas blue color represents the reduced lifetime attributed to fluorescence resonance emission time (FRET) between Alexa Fluor 488 and Alexa Fluor 546 (n = 3–4). c, d Human macrophages (6 × 104/well) (c) and BMDMs (6 × 104/well) from WT and CLEC18 KI mice (d) were incubated with HMW poly (I:C) for 2 h, followed by immunostaining using mAb against CLEC18A/CLEC18A(S339R), TLR3, and EEA1, respectively. Cells were examined by a confocal microscope (TCS-SP5-MP-SMD, Leica). Green color: human CLEC18A and CLEC18A(S339R); red color: TLR3; white color: EEA1. pIC poly (I:C). Scale bar: 10 μM (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001.