FIG 2.

IKKαβ is involved in dsDNA-mediated NF-κBp65 activation in MEFs. (A) Immortalized MEFs derived from wild-type (WT) and IKKα- or IKKβ-deficient mice were stimulated with 10 μg/ml of dsDNA90 for the indicated times. The expression levels of STING, TBK1, TBK1 phosphorylated at Ser172 (p-TBK1), IRF3 phosphorylated at Ser396 (p-IRF3), NF-κBp65 phosphorylated at Ser536 (p-NF-κBp65), NF-κBp65, IKKα, IKKβ, and β-actin were determined by immunoblotting. (B) WT and IKKα- and IKKβ-deficient MEFs were stimulated with 10 μg/ml of dsDNA90 for 6 h, the total RNAs were extracted from these cells, and the expression levels of mRNAs for IL-6 and IFN-β were determined by real-time PCR. Real-time PCR data were normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. *, P < 0.05 (Student's t test). Error bars indicate standard deviations (SD). (C) IKKα-deficient MEFs subjected to RNAi by use of nonspecific (NS) and IKKβ siRNAs were stimulated with 5 μg/ml of dsDNA90 for 6 h, the total RNAs were extracted from these cells, and the expression levels of mRNAs for IL-6, CXCL10, and IKKβ were determined by real-time PCR. Real-time PCR data were normalized to the amount of GAPDH mRNA. *, P < 0.05 (Student's t test). Error bars indicate SD. (D) IKKα-deficient MEFs subjected to RNAi by use of NS and IKKβ siRNAs were stimulated with 5 μg/ml of dsDNA90 for the indicated times, and then the signaling response was determined by immunoblotting using specific antibodies for STING, IRF3 phosphorylated at Ser396 (p-IRF3), TBK1 phosphorylated at Ser172 (p-TBK1), NF-κBp65 phosphorylated at Ser536 (p-NF-κBp65), NF-κBp65, and TBK1, with β-actin serving as a loading control.