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. 2004 Jun 21;199(12):1641–1650. doi: 10.1084/jem.20040520

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Copyright © 2004, The Rockefeller University Press

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Figure 6. — Involvement of IKK-i in IFN-β gene expression in poly(I:C) signaling. (A) Impaired mRNA induction in poly(I:C)-stimulated IKK-i ^−/−TBK1^−/− cells. Immortalized control and IKK-i ^−/−TBK1^−/− EFs were transfected with 10 μg/ml poly(I:C) and incubated for the indicated period. Total RNA was isolated and subjected to Northern blot analysis for the indicated genes. The similar results were obtained from two immortalized EFs. (B) Formation of IRF-3 dimer. Immortalized control (IKK-i ^+/−TBK1^+/−) and IKK-i ^−/−TBK1^−/− EFs were transfected with 10 μg/ml poly(I:C) and incubated for the indicated periods. Whole cell extracts were prepared and subjected to native PAGE. Monomeric and dimeric IRF-3 proteins were detected by Western blotting. The similar results were obtained from two independently established immortalized EFs. (C) NF-κB DNA binding activity. Immortalized IKK-i ^+/−TBK1^+/− and IKK-i ^−/−TBK1^−/− EFs were transfected with 10 μg/ml poly(I:C) for the indicated periods and NF-κB binding was determined by EMSA. Similar results were obtained from two lines of immortalized EFs. *, nonspecific bands. (D) MAP kinase activation. Immortalized IKK-i ^+/−TBK1^+/− and IKK-i ^−/−TBK1^−/− EFs were stimulated with 10 μg/ml poly(I:C) for the times indicated. Whole cell lysates were prepared and blotted with antiphospho-JNK1/2 Ab (phospho-JNK1/2) or antiphospho-ERK1/2 Ab (phospho-ERK1/2). The total amounts of JNK1/2 and ERK1/2 were also determined. One representative experiment from two independent experiments is shown.