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. 2018 Jun 27;9:1491. doi: 10.3389/fimmu.2018.01491

Figure 4.

Figure 4

Cleavage of toll-like receptor 9 (TLR9) is dependent on the amino acid sequence around the cleavage sites. (A) Amino acid sequence of WT, d13, or Ala13 TLR9. Triangles in the figure indicate the cleaved sites. (B) Cleavage pattern of TLR9 with mutation in the cleavage site. FLAG-6xHis tagged-WT or mutant TLR9s were expressed in Ba/F3 cells. FLAG tag was precipitated and C-terminal region of TLR9 was detected by anti-6xHis. β-actin in whole cell lysate (WCL) was detected as an internal control. The intensity of the bands was quantified and shown in the table. (C–E) Response of TLR9s in Ba/F3 cells. FLAG-6xHis tagged-WT or mutant TLR9s were expressed in Ba/F3 cells harboring nuclear factor kappa B (NF-κB)-GFP reporter gene. Expression of TLR9s was detected by flow cytometry (C). Cells were stimulated by TLR9 or TLR2 ligand and activation of NF-κB was monitored as GFP expression by flow cytometry after 24 h incubation (D). Mean fluorescent intensity of NF-κB-GFP was indicated as graph. Cells were stimulated in triplicated wells and the mean SD is shown (E). At least three times of experiments were performed independently (B–E). Data were statistically analyzed by two-way ANOVA with multiple comparisons (D,E). ****p < 0.0001. Abbreviations: ns, not significant; ND, not detected; N/A, not applicable.