The identification of the underlying mechanisms, which regulate the expression levels of the various isoforms, and the elucidation of the physiological relevance for the differential modulation of IRF3 and NF-κB activation will lead to an enhanced understanding of the diverse functions of IKKε in the context of an innate immune response. The Ab against TBK1, phospho-IRF3, phospho-p65 (Ser-536 and Ser-468 specific), and the two different Ab against IKKε (rabbit mAb D20G4 and rabbit polyclonal antiserum recognizing the C-terminus of IKKε) were purchased
from Cell Signaling Technology (Frankfurt am Main, Germany), the anti-FLAG mAb M2 was obtained from Sigma (Taufkirchen, Germany), the anti-myc mAb from Invitrogen (Karlsruhe, Germany), the IRF3 Ab from Epitomics (Burlingame, selleck compound CA, USA), and the actin Ab was purchased from Santa Cruz (Heidelberg, Germany). Poly(I:C) Afatinib cost and blasticidine were obtained from InvivoGen (San Diego, CA, USA). The purification of RNA was performed using the NucleoSpin RNA II kit from Macherey-Nagel (Düren, Germany); cDNA was generated using the First-Strand cDNA Synthesis Kit from GE-Healthcare (München, Germany). Amplification by PCR and ligation into the expression vectors pRK5, pFLAG.CMV2, and pcDNA3.1 myc-His were performed using standard protocols.
Fusion constructs of NAP1, TANK, and SINTBAD with Renilla luciferase were kindly provided by F. Randow (Cambridge, tuclazepam UK) 9. In vitro mutagenesis was performed using the QuickChange kit purchased from Stratagene (La Jolla, CA, USA), following the instructions of the manufacturer. Primers used for PCR and mutagenesis are summarized in Supporting Information Table S1. All constructs were verified by DNA sequencing. To quantify the expression of the different IKKε isoforms, PCR products were cloned into the pCR2.1-TOPO vector using the TOPO-TA cloning kit from Invitrogen. Plasmid DNA was isolated from the resulting colonies and inserts were analyzed by sequencing. HEK293T, MCF7, U937, and THP1 cells were originally obtained from ATCC, 293/TLR3
cells were obtained from InvivoGen. HEK293T, 293/TLR3, and MCF7 cells were grown in DMEM medium, U937 and THP1 cells in RPMI 1640 medium. Both media were supplemented with 10% fetal calf serum and 50 μg/mL each of streptomycin and penicillin. Briefly, 293/TLR3 cells were additionally cultivated with 10 μg/mL blasticidine. HEK293T and 293/TLR3 cells were transiently transfected by standard calcium phosphate precipitation or using FuGene HD (Roche Molecular Biochemicals, Penzberg, Germany) as suggested by the manufacturer. Human PBMC were purified from buffy coats of healthy donors using Ficoll-Hypaque and grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 50 μg/mL of streptomycin/penicillin. The use of buffy coat cells for these experiments was approved by the local Ethics Commission.