Fig. 3: TRIP12–OTUD5 antagonism regulates NF-κB signaling.

a RNA-sequencing analysis. HT1080 cells were transfected with the indicated siRNAs for 72 h and total RNAs were isolated. Three biological replicates were used. b Protein-coding genes whose expression levels are down-regulated (<0.67-fold) by TRIP12 knockdown (siTRIP12 vs. scramble) and restored (>1.5-fold) by OTUD5 double knockdown (siTRIP12+siOTUD5 vs. siTRIP12). c Gene ontology (GO) analysis of genes isolated in (b). The P value stands for the adjusted P-value, which is corrected by the Benjamini-Hochberg method. For gene ontology analysis using DAVID, the raw P values, which were one-sided and not adjusted, were used. d NF-κB signal transduction is down-regulated in TRIP12 knockdown cells. HT1080 cells transfected with the indicated siRNAs for 72 h were treated with TNF-α for the indicated duration, and total lysates were subjected to western blotting. e NF-κB signaling is antagonistically regulated by TRIP12 and OTUD5. Cells were treated as in (d). In the lowest panel, total cell lysates (20 μg) were loaded to detect OTUD5 and OTUD5 band intensities were quantified. f Expression of NF-κB target genes is antagonistically regulated by TRIP12 and OTUD5. HT1080 cells were treated with 0.1 ng/mL TNF-α for 3 h, and RT-qPCR was performed. Four biological replicates were used. g HEK293T cells were transfected with FLAG-HA-OTUD5 plasmids for 48 h, and immunoprecipitation was performed using an anti-FLAG antibody. The association of endogenous TRAF2 with FLAG-HA-OTUD5 was detected. h K63 ubiquitylation of TRAF2 in response to TNF-α stimulation is antagonistically regulated by TRIP12 and OTUD5. HT1080 cells were treated with TNF-α as indicated, and cell lysates were subjected to K63TUBE pulldown to enrich proteins modified with K63-linked ubiquitin chains. i Schematic model. TRIP12 and UBR5 down-regulate the steady-state protein level of OTUD5. Accumulation of OTUD5 under TRIP12/UBR5 deficiency repressed TNF-α-induced NF-κB signaling partly, through deubiquitylation of TRAF2. Source data are provided as a Source Data file.