Figure 7: EGFR forms a signalling complex with IRF3 and TBK1. | Nature Communications

Figure 7: EGFR forms a signalling complex with IRF3 and TBK1.

From: Constitutive and ligand-induced EGFR signalling triggers distinct and mutually exclusive downstream signalling networks

Figure 7

(a) EGFR co-immunoprecipitates with IRF3 in U251EGFR cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR–IRF3 association. (b) Similarly, the EGFR co-immunoprecipitates with TBK1 in U251EGFR cells. When EGF is added, there is a rapid loss of the EGFR–TBK1 association. (c) TBK1 is associated with IRF3 in U251EGFR cells in the absence of EGF. When EGF is added to cells, there is a rapid decrease in TBK–IRF3 association. The TBK1–IRF3 association is also disrupted when Erlotinib is used suggesting that it is EGFR driven. C refers to control vehicle (PBS). (d) EGFR co-immunoprecipitates with IRF3 in MDAMB468 cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR-IRF3 association. (e) Similarly, the EGFR co-immunoprecipitates with TBK1 in MDAMB468 cells. When EGF is added there is a rapid loss of the EGFR–TBK1 association. (f) TBK1 is associated with IRF3 in MDAMB468 cells in the absence of EGF. When EGF is added to cells, there is a rapid decrease in TBK–IRF3 association. The TBK1–IRF3 association is also disrupted when Erlotinib is used suggesting that it is EGFR driven. (g) In contradistinction to IRF3 and TBK1, Shc becomes associated with the EGFR only when EGF is added to U251EGFR cells. (h) About 10% of IRF3 is associated with the EGFR. (i) About 5% of phospho-IRF3 is associated with the EGFR. (j) EGFR associates with IRF3 and TBK1 in the membrane fraction. Membrane, cytosolic or nuclear fractions were prepared from U251EGFR cells. Immunoprecipitation was conducted with IRF3 or TBK1 antibody followed by western blot with EGFR. E-Cadherin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lamin blots were done to test purity of various fractions. Cells were serum starved and cultured in serum-free DMEM overnight for all experiments. The EGF concentration used was 50 ng ml−1. (k) In vitro transcribed and translated EGFR was incubated with GST-IRF3 or GST alone followed by western blot with EGFR. (l) In vitro transcribed and translated EGFR. V, empty vector.

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