Fig. 2

miR-574-5p binds and col-localizes with hTLR8 and mTlr7. Results were representative for 2–3 independent experiments. TCL, total cell lysate; IP, immunoprecipitation; IB, immunoblot. RNA-protein co-immunoprecipitation (co-IP) and fluorescence confocal microscopy were performed as described in the Materials and Methods section. (a) Co-IP assays demonstrating the binding of Dig-miR-574-5p with a truncated hTLR8 but not a truncated hTLR7. His, histidine; PA, protein A. (b) Co-IP assays demonstrating the binding of Dig-miR-574-5p with the full-length hTLR8 but not the full-length hTLR7 and hTLR9. (c) Co-IP assays demonstrating that the binding of Dig-miR-574-5p with a truncated mTlr7 but not a truncated mTlr8. (d) Co-IP assays demonstrating that the binding Dig-miR-574-5p with the full-length mTlr7 but not the full-length mTlr8/9. (e) Localization of transfected miR-574-5p in the endolysosomal compartments. About 1 × 10⁵ HeLa cells were seeded onto 20 mm cell culture plates and grown to 50% confluency. Cells were treated with 3 µg/well Dotap-formulated Cy3-labelled miR-574-5p (Genscript, Nanjing, China) and incubated for 12 h in darkness. The resultant cells were washed four times with PBS and further stained with LysoTracker blue DND-22 (Cat#L7525, Invitrogen, diluted 1:1,000 in Medium) for 10 min before examination under a Zeiss LSM 780 confocal microscope. Scale bar, 20 μm. (f) Confocal fluorescence microscopy demonstrating co-localization of miR-574-5p with hTLR7/8 but not with hTLR4. HeLa cells were transfected with Dotap-formulated Cy3-miR-574-5p (red) stained with either anti-hTLR8 or anti-hTLR7 or anti-TLR4 (green) and DAPI (blue) and visualized under a confocal microscope. The yellow spots in the merged images represented the co-localization of miR-574-5p with the TLRs. A selected cell in the merged images were also digitally magnified (the last column). The scale bars represented 20 μm