We estimated the activation of the transcription factors in our network over time using NCA ( Figure 1B). NCA decomposes a matrix containing gene expression values (E) into a matrix which represents the influence of a transcription factor on a target gene (strength matrix S) and a matrix which contains the transcription factor activities (activity matrix A) [8]. We found that both outputs of NCApredicted factor activities A and regulatory influences Shave added additional insights to gene expression **data** where the underlying regulatory network structure is partially known. Figure 2A and 2B show the estimated activities of our 10 transcription factors. Transcription factor activities clearly showed early-, mid-, and late-phase action in response to LPS. IRF3, NFKB1(p50/p105) and RELA (p60) were activated within 2 hours after the endotoxin was injected. IRF3 activation peaked at 2 hours and returned to its base level at 4 hours. NFKB1 and RELA were also activated early but decreased in activity more slowly. These three factors can induce expression of tumor necrosis factor alpha, which then further activates the NF-B complex [25,26], and could contribute to the extended NF-B activation. JUN and FOS are known to be activated through the JNK pathway [30,31], and had a similar activation profile to NFKB1 and RELA. In contrast, STAT1, STAT3 and CREB1 exhibited a late-phase response. The STAT1 and STAT3 predictions correspond to previous findings that STATs are activated by cytokines transcribed by the NF-B complex [15]. It was surprising that predicted CREB1 activation peaked at four hours, given that previous reports detect phosphorylated CREB at 30 minutes [32]. However, the prediction was the result of late-phase induction of known CREB-dependent gene expression, such as ALAS1 and CEBPD [33,34]. Both STAT6 and MYC were predicted to be somewhat deactivated over nine hours. Deactivation of STAT6 was predicted due to repression of MHC-II class genes which are known to be regulated by STAT6 [35], as well as the expression of SOCS1, which has been reported to lead to deactivation of STAT6 [36]. MYC expression can be decreased through a STAT1-dependent pathway under IFN-γ stimulation conditions [37], and it is possible that the deactivation predicted here depends on STAT1 as well.