Fig. 1

LPS and poly(I:C) elicit divergent responses. a Schematic diagram of the regulatory system of three transcription factors, NF-κB, IRF3 and STAT1/2, activated upon stimulation with LPS or poly(I:C). The synthesis of cytokine IFNβ, mediating autocrine and paracrine signalling, requires activation of both NF-κB and IRF3. Arrow heads = activation, hammer heads = inhibition. b, c Protein levels of the system components in response to LPS or poly(I:C), characterised by western blotting and compared with numerical model simulations. WT MEFs were stimulated with 1 μg/ml LPS or 1 μg/ml poly(I:C). GAPDH and HDAC1 serve as loading controls. Trajectories show averages of 200 independent stochastic simulations; the colour key is located next to protein labels. b Whole-cell extracts were analysed using antibodies against phosphorylated (active) forms of IKKα/β and TBK1, as well as total TBK1, IκBα and A20. Representative experiments out of 2 for LPS and 4 for poly(I:C) are shown. (*) = IKK isoform-dependent phosphorylation sites: p-IKKα Ser176/180, p-IKKβ Ser177/181. c Cytoplasmic and nuclear fractions were analysed using antibodies against total RelA (NF-κB), IRF3 and c-Jun, as well as for phospho-forms (active forms) of IRF3 and c-Jun. Representative experiments out of 2 are shown. d mRNA levels of NF-κB inhibitors, IκBα and A20, in response to LPS, cycloheximide (CHX) with LPS, or poly(I:C). WT MEFs were stimulated with 1 μg/ml LPS in the absence or presence of 5 μg/ml CHX, or with 1 μg/ml poly(I:C). CHX was added 1 h prior to LPS stimulation starting at time = 0. Time profiles of relative mRNA levels were obtained with RT-PCR and then rescaled to absolute numbers using digital PCR measurements. Bars represent means ± s.e.m., n ≥ 2, see Supplementary Note for plots of all replicates compared with model simulations