Figure 6.

Inhibition of NF-κB–dependent transcriptional activity by Shn-2. (A) 293 T cells were transfected with a control pAct or a pAct-Flag-hShn-2 vector, and 2 d later, their cell lysates were prepared. Biotinylated NF-κB and control AP-1 oligonucleotides were absorbed by streptavidin-agarose beads, and then the beads were incubated with cell lysates. The amount of Shn-2 protein in the precipitates was assessed by immunoblotting with anti-Flag mAb. Total cell lysates (10⁶ equivalent/lane) were also run as controls (input). Three independent experiments were performed with similar results. (B) Nuclear extracts were prepared from 293 T cells transfected with pAct-Flag-hShn2 or pCMX-p50 vectors. The extracts were mixed in a certain ratio and then incubated with NF-κB oligonucleotides absorbed with streptavidin-agarose beads. The numbers represent the volume of cell lysates (μl; 1 μl = 5 × 10⁵ cell equivalent). none, nontransfected 293 T lysates. The bound protein was detected by immunoblotting with an anti-Flag mAb for Shn-2 and an anti-p50 mAb for p50 protein. Total cell lysates were also run as controls (input). The position of Shn-2 and p50 are indicated. Three experiments were performed with similar results. (C) 293 T cells were transfected with 25 ng of the 5× NF-κB reporter constructs with the indicated doses of a pAct control vector (Mock) or a pAct-Flag-hShn-2 vector (Shn-2). Additionally, 1 ng pRL-TK vector was added into each transfection as an internal control. 24 h after the transfection, cells were stimulated with 50 ng/ml PMA and 500 nM ionomycin or 10 ng/ml TNF-α for12 h and then assayed for luciferase activity.