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. 2015 Jul 20;5:12300. doi: 10.1038/srep12300

Figure 1. TAK1, but not phosphorylated-TAK1, is required for IKK activation.

Figure 1

A, TAK1 is required for IKK and MAPK activation. TAK1 in RAW264.7 cells was knocked down with lentiviral-based shRNA. After treatment with LPS (100 ng/mL) for the indicated times, cells were lysed and subjected to immunoblot analysis. B, mRNA level is decreased in Map3K7 knocked-down RAW264.7 cells. RAW264.7 cells tranduced with control or shRNA targeting to TAK1 were collected and the total RNA was extracted with TRIzol, reverse transcribed, and analyzed for TAK1 mRNA with Q-PCR. C and D, TAK1 is required for TLR4-mediated TNF and IL-6 production. Control and TAK1-silenced RAW264.7 cells were stimulated with LPS for 2 h, and total RNA was extracted. After reverse transcription, TNF and IL-6 mRNA were analyzed with Q-PCR. E, phosphorylated-TAK1 is not required for IKK activation. TAK1-silenced RAW264.7 cells were reconstituted with Flag-tagged TAK1 wild-type (WT) or T(184,187)A mutant. The cells were incubated with LPS for the indicated times, lysed and kinase activation was analyzed by immunoblotting. The western blots were quantified with densitometry, the relative amount was calculated and the band with the highest intensity was set as 1. The densitometry data was presented as mean values from three independent experiments. Results of Q-PCR are averages ± SD of three separate experiments (*, significant difference).