Fig. 3

OTUD1 was upregulated and participated in inflammatory response after cerebral ischemia. A Luciferase assay of NF-κB activity was used to determine the effect of 15 OUT family members on RIP2-induced NF-κB activity in HEK293 cells. Results are representative of three independent experiments, one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 compared with RIP2 group. B, C Western blot of OTUD1 protein expression in ischemic brain tissue from WT mice after 3 h, 6 h, 12 h, 24 h, 48 h and 72 h MCAO. n = 6 mice per group, one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 compared with sham group. D Representative immunofluorescence image of OTUD1 in mouse ischemic cortex after 24 h MCAO. Double immunofluorescence of RIP2 (red) and Iba1 (microglial cell marker, green) or GFAP (astrocyte marker, green) were performed. Scale bars: 20 μm. E Representative immunofluorescence image of OTUD1 in BV2 cells and primary cultured astrocytes subjected to 90 min OGD and 24 h reoxygenation (OGD/R). Scale bars: 20 μm. F Western blot of OTUD1 in BV2 cells subjected to OGD/R. Results are representative of three independent experiments. two-tailed unpaired t test, *P < 0.05 compared with control group. G Western blot analysis of OTUD1 in primary cultured astrocytes cells subjected to 90 min OGD and 2 h, 6 h, 12 h, 24 h reoxygenation. Results are representative of three independent experiments, one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 compared with control group. H BV2 cells were transfected with either OTUD1 overexpression Flag-OTUD1 vector or control vector for 24 h. Western blot was used to analyse for the protein levels of Flag, p-NF-κB p65 and IκBα in BV2 cells after OGD/R. Results are representative of three independent experiments, two-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05 compared with indicated group. I Scrambled siRNA or OTUD1 targeting siRNA were transfected into the BV2 cells. Western blot was used to analyse for the protein levels of OTUD1, p-NF-κB p65 and IκBα in BV2 cells after OGD/R. Results are representative of three independent experiments, two-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05 compared with indicated group