Fig 2: OTULIN was required for EA to attenuate neuronal injury in focal cerebral ischaemia/reperfusion rats. a Nissl staining was used to observe the morphology of neurons in the ischaemic penumbra of the cerebral cortex. Black and red arrows represent normal neurons and injured neurons, respectively. (b, n = 3) The statistical analysis of injured neurons using Nissl staining. c Immunofluorescence was used to detect TUNEL+ cells and TUNEL+NeuN+ cells. Panels d (n = 3) and e (n = 3) present the quantitative analyses of the TUNEL+ and TUNEL+NeuN+ cells. f NeuN and NF-κB p65 were co-stained to observe the nuclear translocation of neuronal NF-κB p65 using immunofluorescence. ***P < 0.001 versus the Sham group; &&&P < 0.001, &&P < 0.01, and &P < 0.05 versus the tMCAO group; $$$P < 0.001 and $$P < 0.01 versus the tMCAO + EA group; ###P < 0.001 and ##P < 0.01 versus the tMCAO + EA + LV-Scramble group. Scale bar = 100 μm

Fig 3: OTULIN was involved in EA attenuation of astrogliosis in focal cerebral ischaemia/reperfusion rats. a Immunofluorescence was used to stain astrocytes at 24 h and 72 h. (b, n = 6) The mean immunofluorescence intensity of GFAP at 24 h and 72 h was analysed quantitatively. (c, n = 6) The mean longest process of GFAP+ cells was quantitatively analysed. ***P < 0.001 versus the Sham group; &&&P < 0.001, &&P < 0.01, &P < 0.05 versus the tMCAO group; $$P < 0.01 and $P < 0.05 versus the tMCAO + EA group; #P < 0.05 versus the tMCAO + EA + LV-Scramble group. Scale bar (red) = 100 μm, Scale bar (white) = 10 μm

Fig 4: OTULIN is linearly ubiquitinated by LUBAC (HOIP), which is required for the OTULIN–LUBAC/HOIP interaction under an unstressed condition. (A) Workflow of the identification of ubiquitination (GG)-modified peptides of enriched OTULIN. (B) MS/MS spectrum showing the identification of an OTULIN peptide carrying ubiquitination (GG) on lysine (K) 64. Top, MS/MS fragmentation of the peptide (AADEIEK(-GG)EKELLIHER). Bottom, a series of black (b) and blue (y) product ions are detected in the 3-charge MS/MS spectrum, providing high confidence in the identification and localization of the ubiquitin modification. The x axis represents the mass-to-charge (m/z) ratio, and the y axis shows the relative intensity of different peaks. The monoisotopic precursor mass value [M+3H]3+ of the ubiquitinated peptide is shown. (C) Linear ubiquitination of immunoprecipitated HA-OTULIN WT, K64/66R, K34R, and K34/64/66R mutants in HEK293T cells. HEK293T cells were transfected with HA-OTULIN WT or indicated mutants. After 48 h, cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-linear ubiquitin and anti-HA antibodies. (D) Different types of ubiquitination of immunoprecipitated HA-OTULIN WT and K64/66R mutant in HEK293T cells. HEK293T cells were transfected with HA-OTULIN WT or its K64/66R mutant. After 48 h, cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with antibodies for M1-Ub chains, K48-Ub chains, K63-Ub chains, and HA-tag (hemagglutinin tag). (E) Linear ubiquitination of immunoprecipitated endogenous OTULIN in parental and HOIP-KO HEK293T cells. Cell lysates were immunoprecipitated with anti-OTULIN antibody and immunoblotted with indicated antibodies. (F) Linear ubiquitination of immunoprecipitated HA-OTULIN in HEK293T cells. HOIP-KO HEK293T cells were cotransfected with HA-OTULIN and Myc-HOIP WT or its C885S mutant. After 48 h, cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with indicated antibodies. (G) Co-IP analysis of the interaction between Myc-HOIP and HA-OTULIN WT, C129S, Y56F, or K64/66R mutant in HEK293T cells. HEK293T cells were cotransfected with Myc-HOIP and indicated HA-OTULIN constructs in HEK293T cells. After 48 h, cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with antibodies for HA-tag and Myc-tag. (H) Linear ubiquitination of immunoprecipitated Myc-OTULIN in OTULIN-KO MDA-MB-231 cells. Cells were transfected with or without Myc-OTULIN WT, C129S, Y56F, or K64/66R mutants. After 48 h, cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with the indicated antibodies. (+) indicates the samples receiving the specific transfection listed on the left side, and (-) indicates mock controls.

Fig 5: LUBAC (HOIP) and OTULIN play opposing roles in genotoxic NF-κB activation. (A) HOIP-KO HEK293T cells were reconstituted with an HOIP WT or C885S mutant. After transfection for 48 h, cells were treated with TNFα (T, 10 ng/mL, 15 min) or Etop (E, 10 μM, 2 h). Whole-cell lysates were collected later and subjected to immunoblotting with antibodies as indicated. (B) Gel shift analysis of NF-κB activation by using Igκ probe in MDA-MB-231 cells pretreated with or without LUBAC inhibitor gliotoxin (1 μM) for 24 h and subsequently treated with CPT (10 μM, 2 h) or Dox (2 μg/mL, 2 h). Total cell lysates were analyzed by immunoblotting with antibody against α-tubulin. (C) Parental and OTULIN-KO MDA-MB-231 cells were exposed to IR (10 Gy, +) or were not (-), then were left in the incubator for 2 h. NF-κB activation and protein expression levels of cells were analyzed by gel shift assay and immunoblotting with antibodies as indicated. (D) OTULIN-KO HEK293T cells were reconstituted with OTULIN WT or C129S mutant. After transfection for 48 h, cells were treated with TNFα (T, 10 ng/mL, 15 min) or Etop (E, 10 μM, 2 h). Whole-cell lysates were collected later and subjected to immunoblotting with antibodies as indicated. (E) Immunoblotting analysis of protein expression levels in parental (P) and OTULIN-KO MDA-MB-231 cells treated with Dox (2 μg/mL) for indicated times. (F) Parental, OTULIN-KO, and OTULIN-reconstituted HEK293T cells were cotransfected with κB-Fluc/hRluc-TK reporter constructs. After transfection for 24 h, cells were treated with or without Etop (10 μM, 6 h), and cell extracts were prepared to determine luciferase activity. The activities of firefly and Renilla luciferases were evaluated sequentially from a single sample in the dual-luciferase reporter assay. Results were plotted as firefly normalized to Renilla luciferase activity. The relative luciferase activities compared with the control (lane 1) are shown as mean ± SEM from the experiments performed in triplicate. Two-way ANOVA followed by Tukey’s posthoc test was used to determine statistical significance for multiple comparisons. ***P < 0.001; ****P < 0.0001; ns, not significant.