Figure 5

TLR4 does not mediate gene induction caused by Nm . (A) Expression profile of PRRs in HIBCPP cells. Qualitative RT-PCR was performed to determine the expression of the indicated PRRs, co-receptors, and adaptor molecules in HIBCPP cells (upper panel) and purified human monocytes (lower panel). Expression of β-actin and GAPDH was analyzed as control. The size of relevant marker nucleic acids is indicated. (B) Semi-quantitative RT-PCR was performed to measure activation of nfkbiz, il6, and il8 after 4 h treatment with either Nm MC58siaD^-, “ultrapure” LPS, or Fsl-1 at the indicated multiplicity of infection (MOI) or concentrations, respectively. Control experiments were performed in absence of a stimulus. PCR reactions were analyzed after the cycle numbers indicated at the top of the panels. (C) HIBCPP cells were challenged with 10 μg mL^-1 “ultrapure” LPS or 100 ng mL^-1 Fsl-1 for the indicated time. Expression of IκBζ protein was analyzed by immunoblot analysis. Detection of β-actin protein levels served as control. (D) Semi-quantitative RT-PCR was performed to determine activation of nfkbiz and il6 in primary human monocytes after treatment with 1 vg mL^-1 “ultrapure” LPS. Inhibition experiments were performed with an antibody against human TLR4 or with an isotype control, respectively.