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. 2011 Jul 18;6(7):e20659. doi: 10.1371/journal.pone.0020659

Figure 7. The NRAGE peptide inhibits some cytoplasmic BMP MAPK pathway members, binds to NRAGE and XIAP, and inhibits downstream activation of NF-κB and IKK.

Figure 7

(A) Western blot of endogenous expression of NRAGE, XIAP, and TAK1 in P19 cells after XIAP or NRAGE immunoprecipitation in lyses and NRAGE and Beta-Actin expression of total cell lysates from cells that underwent BMP-4 treatment with and without the NRAGE peptide compared to untreated cells. (B) CFP:YFP FRET assessment of CFP or YFP tagged NRAGE peptide, full-length NRAGE, and XIAP in fixed 293T cells. (C) Western blotting shows that activation of IKK by BMP-4 in HEK293 cells is inhibited in cells cultured with the NRAGE peptide. (D) NF-κB activation is inhibited in HEK293 cells cultured with the NRAGE peptide compared to HEK293 cells that did not receive the peptide as assessed by luciferase assay. Treatments were 10 ng/ml BMP-4, 10 nM NRAGE peptide, and 6 µM EndoPorter delivery reagent where indicated. **P<0.005. C-NR, CFP-NRAGE; Y-NR, YFP-NRAGE; C-X, CFP-XIAP; Y-X, YFP-XIAP; pep-C, NRAGE peptide-CFP; pep-Y, NRAGE peptide-YFP. (E) GFP control P19 cells and the F10R3 NRAGE repeat domain P19 cells treated with and without 10 ng/ml BMP-4, then cell membranes and cytoplasm ran seperatly to see the affects of XIAP and NRAGE expression. Beta Actin was used as loading control.