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. Author manuscript; available in PMC: 2011 Oct 19.
Published in final edited form as: J Biol Chem. 2006 Dec 8;282(6):3918–3928. doi: 10.1074/jbc.M608867200

FIGURE 1. Defining the region of TAK1 that interacts with TAB2 and TAB3.

FIGURE 1

A, Schematic diagram of TAK1 constructs used in this study. The N-terminal region of TAK1 containing the kinase domain is highlighted in black. The C-terminal region is in white. The numbers for mouse TAK1 represent the N- and C-terminal residues of the respective construct. B, The region between residues 479–547 of TAK1 is required to bind TAB2. HEK293 cells were transiently transfected with empty vector (−) or co-transfected with FLAG-TAB2 in the absence or presence of the indicated FLAG-TAK1 constructs. After 36 h, cells were harvested and lysates were immunoprecipitated with anti-TAB2 and immunoblotted with anti-FLAG (top). Expression of the proteins in the cell lysates was determined by immunoblotting with anti-FLAG (bottom). C, TAB2 binds a motif between residues 479–547 within the C-terminal region of TAK1. HEK293 cells were transiently transfected with empty vector (−) or with the indicated FLAG-TAK1 constructs. After 36 h, cells were harvested and lysates were mixed with glutathione agarose beads coated with either GST (G) or GST-TAB2 (T). Bound proteins were determined by immunoblotting with anti-FLAG (top) and expression of the proteins in the lysates was determined by immunoblotting with anti-FLAG (bottom). D–E, Residues 521–545 are not essential for TAB2 interaction. HEK293 cells were transiently transfected with empty vector (−) or co-transfected with FLAG-TAB2 in the absence or presence of the indicated FLAG-TAK1 constructs and immunoprecipitation with anti-TAB2 (D) or a GST pull down (E) was performed essentially as described above. F, TAB3 binds to a similar region on TAK1. HEK293 cells were transiently transfected with empty vector (−) or with the indicated FLAG-TAK1 constructs. After 36 h, cells were harvested and lysates were mixed with glutathione agarose beads coated with either GST (G) or GST-TAB3 (T). Bound proteins were determined by immunoblotting with anti-FLAG (top) and expression of the proteins in the lysates was determined by immunoblotting with anti-FLAG (bottom).