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. 2009 May 22;284(21):14136–14146. doi: 10.1074/jbc.M109.000414

FIGURE 4.

FIGURE 4.

The overexpression of TBK1 and IKKε leads to autophosphorylation and transphosphorylation of Ser-172. A, wild type (WT) and two different catalytically-inactive FLAG-tagged mutants of TBK1 (TBK1-(K38A) and TBK1[D157A]) were expressed in HEK293 cells. Cell extract (40 μg protein) was then subjected to SDS-PAGE and immunoblotted using anti-Ser(P)-172 TBK1 and anti-FLAG. B, FLAG-WT-TBK1 was transfected into HEK293 cells. 24 h later the cells were treated without (–) or with (+)1 μm BX795 for the times indicated. Cell extracts (40 μg protein) were then immunoblotted using anti-FLAG and anti-Ser(P)-172 TBK1 as in A. C, a vector encoding FLAG WT-TBK1 was transfected into HEK293 cells, then incubated for 1 h without (–) or with (+)1 μm BX795. TBK1 was then immunoprecipitated from 0.1 mg of cell extract protein with anti-FLAG (washed) and assayed for activity using the IκBα peptide substrate (mean ± S.E., n = 4; *, p < 0.001), D, a vector encoding FLAG-tagged TBK1-(K38A) was co-expressed in HEK293 cells with an empty vector (–), a vector encoding GST (+), or a vector encoding GST-WT-TBK1 (TBK1) GST-WT-IKKε (IKKε). The cells were lysed, and 40 μg of cell extract protein was immunoblotted (WB) using anti-Ser(P)-172 TBK1/IKKε or anti-FLAG as in A (upper two panels). A further 0.5 mg of cell extract protein was incubated for 1 h at 4 °C with 0.01 ml of glutathione-Sepharose beads with continuous mixing. The beads were collected by centrifugation and washed three times in lysis buffer, and bound proteins were released with SDS and immunoblotted with anti-GST or anti-FLAG antibodies (lower two panels). E, wild type and catalytically-inactive mutants of FLAG-TBK1 and FLAG-IKKε were expressed in HEK293 cells, and the lysates were immunoblotted using anti-FLAG as in A. F, WT-FLAG-IKKε was expressed in HEK293 cells. 24 h later the cells were treated with 1 μm BX795 for the times indicated. Cell extracts (40 μg protein) were then immunoblotted using anti-FLAG as in A. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.