Fig. 3. TBK-1 interacts with and functions in STAT3 Ser727 phosphorylation.

a iBMDMs were seeded at 4 × 10⁴ cells per well in triplicate 24 h prior to pretreatment with kinase inhibitors (Y-axis; 500 nM) for 30 min. MitoSOX was added for 10 min prior to challenge with LPS (100 ng/ml; 0, 1, 2 and 4 h). LPS-induced production of superoxide by mitochondria was analysed by measuring oxidised MitoSOX fluorescence at 580 nm. See also Supplementary Fig. 1. Cell lysates from LPS stimulated BMDMs (0–120 min) were immunoprecipitated with b anti-TRAF6 and c anti-STAT3 antibodies. TBK-1 interaction with TRAF6 and STAT3 was identified by immunoblot with anti-TBK-1 and anti-TBK-1 pS172 antibodies respectively. b, c Data represents three independent experiments with similar results. d TBK-1-deficient and WT macrophages were treated with LPS or IFNα for indicated times and STAT3 phosphorylation assayed in cellular lysates by immunoblot with indicated antibodies. TBK-1 expression was ascertained by immunoblot with anti-TBK-1 antibody. e BMDMs were pretreated with the TBK-1/IKKε (1 μM) inhibitor BX-795 for 60 min prior to LPS stimulation for indicated times. STAT3 and TBK-1 phosphorylation was observed by immunoblot with indicated antibodies. DMSO was added to lanes 13–14 as vehicle control. Immunoblot results are representative of three independent experiments and data presented (d, e) as mean ± S.E.M.