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John H. Fingert, Ben Roos, Frances Solivan-Timpe, Melissa Humbert, Seongjin Seo; Identification Of Proteins That Interact With TANK binding kinase 1 (TBK1). Invest. Ophthalmol. Vis. Sci. 2012;53(14):3848.
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Duplication of the TANK-binding Kinase 1 (TBK1) gene has been associated with familial normal tension glaucoma (NTG). The purpose of this research is to identify the proteins that interact with TBK1 as additional candidates for causing NTG
We generated a plasmid that contains the full-length TBK1 cDNA with two epitope tags (FLAG and S-antigen) that is expressed under the control of CMV promoter sequences. HEK293 cells were transfected with this plasmid and stable lines expressing epitope-tagged TBK1 were generated. TBK1 and interacting proteins were isolated from cell extracts by tandem affinity purification, first using the FLAG epitope then using the S-protein epitope. Isolated proteins were analyzed with SDS-polyacrylamide gel electrophoresis and were identified using mass spectrometry.
A total of five prominent protein bands were identified. One of the bands represents the tagged TBK1 and the other 4 bands represent TBK1 interacting proteins. Mass spectrometry identified these proteins as 5-azacytidine induced protein 2 (AZI2), TRAF family member-associated NFKB activator (TANK), TANK-binding kinase 1 binding protein 1 (TBKBP1), and TANK binding Kinase 1 (TBK1).
We have identified four unique proteins interact with TBK1 in HEK293 cells. One of the proteins is TBK1, suggesting that TBK1 may interact with itself to form homo-oligomers. The other three identified proteins (TANK, TBKBP1, and AZI2) were previously known to associate with TBK1, however, these experiments provide additional support for this interaction. The identity of these TBK1 interacting proteins will help elucidate the mechanism by which defects in TBK1 and the NF-KB pathway contribute to glaucoma pathogenesis. Furthermore, the genes encoding each of these proteins represent new candidates for causing NTG.
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