Study shows immunecells have a backup mechanism frontiersin
THP1-Dual cells were seeded in 450 µl complete RPMI. Then, 150 µl of a cycloheximide dilution in complete RPMI were added, resulting in a final concentration of 10 µg/ml. After 4, 8, 12, 16, and 20 h, the cells were lysed and protein expression of IKKϵ was determined by SDS-PAGE and immunoblotting.For the analysis of TBK1-dependent IKKϵ ubiquitination, stable IKKϵ-GFP-expressing cells were generated by transducing IKKϵ-deficient THP1-Dual cells with IKKϵ-GFP-encoding lentivirus.
To detect non-covalent interactions of TBK1 with TANK, NAP1, SINTBAD, and IKKϵ, tag-based co-IP was performed. Flag-tagged TBK1 WT, TBK1 L693A, TBK1 K694E, or TBK1 L704A was stably expressed in TBK1-deficient THP1-Dual cells by lentiviral transduction followed by puromycin selection. Cells were washed with PBS twice and lysed in 700 µl TAP lysis buffer supplemented with protease and phosphatase inhibitor by incubation on ice for 30 min with regular vortexing.
Although TBK1 is well-known for its essential function in innate immune responses in mice, loss of human TBK1 surprisingly is not associated with an increased susceptibility to severe infections . Based on the observation from this recent study, we aimed at further investigating the consequences of TBK1 deletion for antiviral defense of human monocytic cells, essential players in innate immune recognition and important inducers of type I IFN upon viral infection.
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