To establish NF-κB–responsive luciferase reporter cell lines, HEK293T cells were transduced with a lentiviral vector containing a luciferase reporter gene under control of three NF-κB–binding sites. Single cell clones of transduced cells were established by limiting dilution, and inducible NF-κB activity was confirmed by transfection with various TLR adaptor proteins. Stable reporter cell lines expressing GyrB fusion proteins were established by viral transduction (MyD88-GyrB and TIRAP-GyrB) or lipofectamine transfection (TRAF6-GyrB), followed by antibiotic selection. Stably growing cells were cloned by limiting dilution, and clones that showed CM-mediated NF-κB activation were selected for further experiments.

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