Fig. 3.

Nuclear factor kappa B (NF‐κB) is required for Mycoplasma pneumoniae lipid‐induced tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β secretion. RAW264.7 cells (2 × 10⁶ cells/ml) were cultured and separately preincubated with anti‐Toll‐like receptor (TLR‐2) or anti‐TLR‐4 neutralizing antibody (10 μg/ml) for 1 h on coverslips in a 24‐well plate, following treatment with lipids for 6 h. Immunofluorescence was used to detect the translocation of NF‐κB (a) and data were quantified by counting more than 50 cells in triplicate (b) or the cell lysates were subjected to Western blot analysis using anti‐inhibitor NF‐κB (IκB) antibody (c). (d) Bone marrow‐derived macrophages (BMDMs) were pretreated with 5~10 μmol/l of the NF‐κB inhibitor Bay 11‐7082 for 40 min. Later, enzyme‐linked immunosorbent assay (ELISA) was used to determine the concentration of TNF‐α and IL‐1β before and after treatment with lipids. The data represent three independent experiments [mean ± standard error of the mean (s.e.m.)]; *P < 0·05 denotes significant difference between the compared groups.