Fig. 2.

Delayed activation of NF-κB signaling in cells simultaneously stimulated with both agonists for TLR7 and TLR9. (A) RAW264.7 cells were stimulated with either an agonist for TLR7 or TLR9 or both. The cell lysates were extracted, and the changes in expression levels of TLR signaling downstream molecules were assessed using Western blot analysis. Bar graphs indicate the results of the relative densitometry compared with β-actin protein. (B) Nuclear translocation of p65 was identified within cytoplasmic (CE) and nuclear fractions (NE) by Western blotting against p65. (C) RAW264.7 cells were transfected with the NF-κB-luc reporter gene. The transfected cells were treated with either an agonist for TLR7 and TLR9 or both for 4 h (1st), followed by washing with PBS and incubation in culture media for 12 h. Subsequently, the cells were restimulated with TLR agonists for 1 h (2nd), and the NF-κB promoter activity was measured through luciferase assay. Significant differences were indicated as **P < 0.01, ***P < 0.001, and n.s., not significant (P < 0.05).