Fig 2: Interaction with E. coli O111:B4 lipopolysaccharide and immunomodulatory activity of PN5. (A) CD analysis of the binding structure of AMPs with lipopolysaccharide (LPS); (B) effect of PN5 (2, 4, and 8 μM) on LPS-induced TNF-α production. The concentrations of TNF-α in the culture supernatants were determined by ELISA. (C) Western blot of TNF-α, TLR4, and β-actin expression. LPS-pretreated RAW 264.7 cells were cultured with PN5 at different concentrations for 24 h. (D) Graphs were generated by quantifying the Western blots using ImageJ software. (E) Immunofluorescence staining showed that PN5 inhibited TNF-α expression induced by E. coli LPS. (F) The Western blot was performed for NF-κB and MAPK signaling activation in RAW264.7 cells. Intracellular NF-κB (p65), IκBα, p38, ERK, and JNK signals were confirmed. Values represent the mean ± SEM from three individual experiments. **, P < 0.01, and ***, P < 0.001, by unpaired t test.