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. 2007 May 14;404(Pt 2):179–190. doi: 10.1042/BJ20061704

Figure 5. TAK1 is required for MDP–NOD2/RIP2 signalling.

Figure 5

All experiments were carried at least twice with similar results. (A) Control HEK-293 cells or cells depleted of TAK1 by transfection with an shRNA were generated as described in the Experimental section. The cells were lysed, TAK1 immunoprecipitated as described in the legend to Figure 4, and the pellet extracted in SDS and immunoblotted with anti-TAK1 antibodies. (B) HEK-293 cells were co-transfected with 0.1 μg of DNA encoding 3× κB luciferase reporter construct (see the Experimental section), 0.1 μg of pTK-RL plasmid DNA (encoding Renilla luciferase) plus 0.8 μg of empty pCMV5 vector (black bars) or plasmids encoding GST-NOD2 (black bars) or HA-tagged WT-RIP2 (white bars). After 48 h, the cells were lysed and NF-κB-dependent luciferase reporter-gene expression was analysed. The error bars show the S.D. between duplicate experiments. (C) Experiments were performed as described in the legend to Figure 3(A), using 1 ng of DNA encoding HA–NOD2 or empty-vector DNA for transfection of control HEK-293 cells or cells depleted of TAK1. After 24 h, cells were incubated without (white bars) or with (black bars) 1 μM MDP. After 24 h the cells were lysed and NF-κB-dependent luciferase reporter-gene expression was analysed. The error bars show the S.D. between duplicate experiments. (D) Experiments were performed as in (C). At 1 h before stimulation with (black bars) or without (white bars) MDP, the cells were incubated without (control) or with 1 μM (5Z)-7-oxozeaenol, a TAK1 inhibitor. The error bars show the S.D. between duplicate experiments. (E) WT (+/+) or TAK1−/− (−/−) mouse embryonic fibroblasts were co-transfected with 0.5 μg of DNA encoding 3× κB luciferase reporter construct (see the Experimental section), 0.5 μg of pTK-RL plasmid DNA (encoding Renilla luciferase), plus 4 μg of empty pCMV5 vector (grey bars) or plasmids encoding GST–NOD2 (black bars) or HA-tagged WT-RIP2 (white bars) using the Amaxa Nucleofection MEF2 kit according to the manufacturer's instructions. After 48 h the cells were lysed and NF-κB-dependent luciferase reporter-gene expression was analysed. The error bars show the S.D. between duplicate experiments.