Role of NF-κB activation and MAP kinase phosphorylation in the LPS sensitivity phenotype of DAF^−/− mice. (A) Western blot analysis showing the time course of IκBα phosphorylation in wild-type (WT) and DAF^−/− mouse spleens after LPS challenge. Each time point represents an individual mouse. (B) Western blot analysis of IκBα phosphorylation in the spleens of 4 WT and 4 DAF^−/− mice at 30 minutes after LPS challenge. (C) Western blot analysis of IκBα levels in the spleens of 4 WT and 4 DAF^−/− mice at 60 minutes after LPS challenge. (D) Effect of C5a (50 nM) on LPS (100 ng/mL)–induced activation of an NF-κB luciferase reporter gene and TNF-α production in RAW264.7 cells. Cells were transiently transfected with the reporter gene plasmid together with a human C5aR cDNA construct. NT, No treatment. (E) Western blot analysis showing the time course of ERK1/2 phosphorylation in WT and DAF^−/− mouse spleens after LPS challenge. Each time point represents an individual mouse. (F) Western blot analysis of JNK phosphorylation in the spleens of 4 WT and 4 DAF^−/− mice at 60 minutes after LPS challenge. Relative amount of each protein was expressed as the ratio between the protein and β-actin signals on Western blots.