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The reduced interferon response was not due to drug-induced toxicity (Figure?6G). Next, we tested the effects of Plk inhibition in virally infected mice. BI 2536 exhibits good tolerability in mice (Steegmaier et?al., 2007) and humans (Mross et?al., 2008) and is currently in phase II clinical trials as an antitumor agent in several cancers (Strebhardt, 2010). Given its efficacy and safety in?vivo, we tested whether BI 2536 would also affect the response to viral infection in animals. In mice infected with VSV, BI 2536 strongly suppressed mRNA production in popliteal VX-770 in vivo lymph nodes for type I IFNs (Ifnb1, Ifna2) and Cxcl10 but did not affect Cxcl1 mRNA induction (all compared to vehicle control; Figures 6H and S6D). Concomitantly, VSV replication in the lymph node rapidly increased as reflected by elevated VSV RNA levels ( Figure?6I), comparable to the observed phenotype of VSV-infected Ifnar1?/? mice ( Iannacone et?al., 2010). Because in the VSV model used here type I IFNs are produced by both infected CD169+ subcapsular sinus macrophages and pDCs ( Iannacone et?al., 2010), we cannot distinguish whether Ribonucleotide reductase Plk inhibition affects macrophages, pDCs, or both. Nevertheless, our results confirm the physiological importance of Plks in the host antiviral response in both ex?vivo primary MLFs and in?vivo mouse lymph nodes. We next sought to discover the signaling pathways between Plks and antiviral gene transcription. We used microwestern arrays (MWAs) (Ciaccio et?al., 2010) to measure changes in the phosphorylation and protein levels of 20 and 6 TLR pathway proteins, respectively, in BMDCs at each of 12 combinations of four time points (0, 20, 40, 80?min after LPS stimulation) and three perturbations (vehicle control, BI 2536, and negative control JNK inhibitor SP 600125) (Table CH5424802 S6). Although LPS stimulation alone led to the expected changes (e.g., early peak of phosphorylation for ERK1/2, p38, and Mapkapk2 and rapid degradation of I��B��; Figure?7A), BI 2536 surprisingly did not cause any significant changes (Figures 7A, S7A, and S7B). We therefore hypothesized that Plks could affect previously unrecognized regulators of IFN-inducing pathways and/or known regulators with no existing antibodies to specific phosphosites. Next, we used SILAC-based unbiased phosphoproteomics (Figure?7B, top) (Vill��n and Gygi, 2008) to compare the levels of?phosphotyrosine, -threonine, and -serine peptides following stimulation with LPS (for 30 or 120?min) in BMDCs pretreated with BI 2536 versus those treated with vehicle (DMSO). We identified and quantified 5,061 and 5,997 phosphopeptides after 30?and 120?min, respectively, for a total of 10,236 individual phosphosites (Figure?7B and Table S6). BI 2536 substantially affected the TLR phosphoproteome, leading to a significant (p?