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Fig. 2
Regulation of zebTLR9 and zebTLR21 activation by UNC93B1. HEK293 cells were cotransfected with expression vectors for FLAG-tagged WT and mutant zebTLR9 and zebTLR21, Myc-tagged UNC93B1, and NF-κB luciferase reporter gene as indicated. (A) Cellular localizations of proteins were visualized by immunofluorescence staining. Colocalizations of UNC93B1 with zebTLR9 and zebTLR21 were quantified by calculating the Pearson correlation coefficient of the signaling intensities of the two proteins. (C) Protein binding was measured by immunoprecipitation followed by immunoblotting. (D) Cells were treated with 3 µM CpG-ODN, after which relative luciferase activity was measured. Data are mean ± SD (n = 3). Immunoblots shown represent expression levels of WT and acidic amino acid residue-mutated zebTLR9 and zebTLR21. (B) Sequence alignment of the juxtamembrane regions of different human and zebrafish TLRs. The acidic amino acid residues in intracellular TLR regions are shown in red.