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. 2022 Aug 8;13(8):694. doi: 10.1038/s41419-022-05145-5

Fig. 2. OTUD1 downregulates inflammatory cytokine-induced canonical NF-κB activation.

Fig. 2

a The enhanced ubiquitination and recruitment of NF-κB signaling factors to TNFR complex I. Parental and OTUD1−/−-HeLa cells were stimulated with 1 μg/ml FLAG-TNF-α for the indicated periods, and cell lysates and anti-FLAG immunoprecipitates were subjected to immunoblotting with the depicted antibodies. b K63-linked polyubiquitination of NF-κB activators is enhanced in OTUD1−/− cells. Parental and OTUD1−/−-HeLa cells were stimulated with 20 ng/ml TNF-α for the indicated periods, and then pulled down by K63-TUBE. Samples were subjected to immunoblotting with the indicated antibodies. c The enhanced IL-1β-induced NF-κB activation in OTUD1−/− cells. Parental and OTUD1−/− cells were stimulated with 1 ng/ml IL-1β, and analyzed as in a. d, e Alterations of K63- and M1-linked ubiquitination of NF-κB activators in OTUD1−/−-HeLa cells. Parental and OTUD1−/− cells were stimulated with 1 ng/ml IL-1β for the indicated periods, and pulled down by K63-TUBE (d) or M1-TUBE (e). Samples were immunoblotted with the indicated antibodies. f, g RNA-seq analysis. Parental and OTUD1−/−-HeLa cells were stimulated with 10 ng/ml IL-1β for 1 and 3 h. The cells were lysed and subjected to a transcriptome-wide expression analysis using their extracted total RNA. The principal component analysis (f) and heatmap analysis of significantly varied 34 mRNAs, using the cut-off value of 10 (g) was performed. h The enhanced inflammatory responses in OTUD1−/−-HeLa cells. Taking the cut-off value of 3.0, pathway analyses of 175 up-regulated genes were performed by MSigDB Hallmark 2020, and are listed in ascending order of P-value.