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. 2015 Aug 23;98(6):963–974. doi: 10.1189/jlb.2VMA0515-229RR

Figure 7. Pellino-3b acts within the MyD88-IRAK1-TRAF6-TAK1 and TRIF-TBK1 signaling axes but does not affect p65-induced NF-κB reporter activation.

Figure 7.

(A) HEK293T cells were transfected with pELAM-Luc (0.4 μg/well) and pTK-RL (20 ng/well) plus the indicated amounts of pcDNA3-AU1-MyD88 or pcDNA3-TRIF or expression vectors encoding IRAK1, TBK1, TRAF6, TAK1, or p65 (10 ng/well each), with or without p-Super-Flag-Pellino-3b (0.2 and 0.8 μg/well; 2 left panels in the first row of A; 0.8 μg/well for all other panels of A). After recovery for 48 h, cell lysates were assayed for firefly vs. Renilla luciferase activities. Relative luciferase units of firefly Luc were normalized to those for Renilla Luc, and the data were presented as the fold induction compared with the values in cells transfected with pcDNA3, pELAM-Luc, and pTK-RL taken as 1. (B) HEK293T cells were plated in 6-well plates and transfected with pcDNA3, pRK5-IRAK1, or pSuper-TBK1 (1 μg/well), along with pcDNA3 or Flag-Pellino-3b (1 μg/well). After recovery for 48 h, cell lysates were analyzed by Western blotting with anti-IRAK1, anti-p-TBK1, anti-TBK1 and anti-Flag Abs. The data of a representative (n = 3) experiment are shown.