To identify novel interactors of TBK1, we generated stable RAW264.7 cell lines that express a GS-TAP-tagged version of TBK1 and purified the TBK1 protein complex by tandem affinity purification (Figure 1A and B) (Burckstummer et al, 2006).
GS-TAP-tagged DDX3X, isolated by affinity purification using rabbit IgG agarose, was also recruited to the enhanceosome region upon L.
monocytogenes infection (Figure 5C).
This recruitment was specific because a control region (Figure 5A) could not be amplified by PCR under these conditions.
This suggests that DDX3X exerts a direct effect on the IFN-β promoter.
Tandem affinity purification using the GS-TAP cassette was performed as described previously (Burckstummer et al, 2006).
The DEAD-box helicase DDX3X is a target of TBK1.
(A) Schematic representation of the tandem affinity purification protocol: GS-TAP-tagged TBK1 expressed in RAW264.7 cells was purified using rabbit immunoglobulin G (IgG) agarose and eluted by tobacco etch virus (TEV) protease cleavage.
Next, the remaining complex was purified using streptavidin agarose and eluted by boiling in SDS sample buffer.
(B) The final TAP eluate was separated on an SDS–PAGE and stained by silver staining.
Protein complex composition was analysed by LC-MSMS.
The position of several complex components is depicted next to the region where it was identified.