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. 2017 Nov 17;37(1):19–38. doi: 10.15252/embj.201696164

Figure EV2. LPS but not EGF increases TBK1 activation loop site phosphorylation in MEFs; TBK1 is not required for amino acid stimulated mTORC1 signaling; ectopic TBK1 overexpression suppresses mTORC1 signaling (related to Fig 2).

Figure EV2

  1. EGF fails to increase TBK1 S172 phosphorylation in MEFs. Cells were serum deprived (20 h) and stimulated −/+ EGF [25 ng/ml] or LPS [100 ng/ml] for the times indicated. Whole‐cell lysate (WCL) was immunoblotted as indicated. SE: short exposure; LE: long exposure.
  2. EGF fails to increase TBK1 S172 phosphorylation in HEK293/TLR3 cells. Cells were serum deprived (20 h) and stimulated −/+ EGF [25 ng/ml] or poly(I:C) [50 ng/ml] for the times indicated.
  3. TBK1 is not required for amino acid‐stimulated mTORC1 signaling. TBK1+/+ and TBK1−/− MEFs were deprived of amino acids by incubation in D‐PBS + glucose + dialyzed FBS [10%] (60 min). Amino acids were added back by incubating the cells in DMEM/FBS [10%] (30 min).
  4. Cellular overexpression of TBK1 inhibits mTORC1 signaling. HEK293 cells were co‐transfected with HA‐S6K1 together with Flag‐TBK1, serum‐starved (20 h), pre‐treated with Ku‐0063794 [1 μM] (30 min), and stimulated −/+ EGF [25 ng/ml] (30 min). HA‐S6K1 was immunoprecipitated, and IPs and WCL were immunoblotted (IB) as indicated.