Fig. 2. Oligomeric NP interacts with and activates TLR2 and TLR4.

a Bone marrow-derived macrophages (BMDMs) were treated with recombinant NP (rNP; 100 μg/ml) for 0–120 min or b with rNP (12.5–100 μg/ml) for 30 min. After the incubation period, the activation of the NF-κB and MAPK pathways was detected by western blotting. c BMDMs were treated with rNP (12.5–100 μg/ml) for 20 h, and the levels of cytokines and chemokines in the cell culture supernatant were measured by ELISA. d HEK293 cells expressing TLR2, TLR3, TLR4, TLR5, TLR7, or TLR9 were incubated with PBS, rNP (12.5–100 μg/ml), or the corresponding ligands (1 μg/ml PAM3, 1 μg/ml poly I:C, 100 ng/ml LPS, 1 μg/ml flagellin, 1 μg/ml R848, or 1 μg/ml CpG oligodeoxynucleotide) for 20 h. After the incubation period, the absorbance was recorded at 630 nm. e BMDMs were treated with rNP (0–30 min), followed by immunoprecipitation (IP) with anti-Toll-like receptor (TLR) 2, anti-TLR4, and anti-NP antibodies. TLR2, TLR4, and NP in the coprecipitate were detected by western blotting. f rNP (6.25–100 μg/ml) was added to each well coated with recombinant TLR (rTLR) 2 (2 μg/ml) and rTLR4 (2 μg/ml). After sequential incubation and washing, NP bound to rTLRs was detected by ELISA. g TLR2 KO, TLR4 KO, and TLR2/4 DKO BMDMs were treated with rNP (100 μg/ml) for 20 h. After the incubation period, the cytokine and chemokine levels in the cell culture supernatant were determined by ELISA. HEK293 cells expressing TLR4 were cultured with PBS, LPS, h 100-kDa size-fractionated rNP (3.125–25 μg/ml), i the rNP R416A mutant (12.5–100 μg/ml), j or 25 °C, 4 °C, and –80 °C-incubated rNP (12.5–100 μg/ml). After 20 h, the absorbance was recorded at 630 nm. Data are presented as the mean ± SD from triplicate culture wells. ***p < 0.001, **p < 0.01, *p < 0.05