Fig. 3

SENP6 downregulation decreased NF-κB signalling pathway activity in primary cultured microglia subjected to OGD/R. A Representative immunoblots showing the phosphorylation of IKKα/β, IκBα and p65 in microglia infected with adenovirus encoding vector, SENP6, sh. NC and sh. SENP6 following OGD/R or not. B–E Quantification of the p-IKKα/β, p-IκBα and p-p65 proteins in A. F Immunoblots detected NF-κB p65 in cytoplasmic and nuclear fractions of primary microglia, as well as in whole-cell lysates. G, H Quantitative analysis of NF-κB p65 protein levels in the cytoplasm and nuclear extracts of microglia in F. I The p65 transcriptional activity was confirmed by a Dual-Luciferase reporter assay in HEK293T cells transfected with a plasmid expressing SENP6 or sh. SENP6. J The change in p65 transcriptional activity in HEK293T cells with increasing SENP6 doses. K Immunofluorescence staining determined the subcellular localization of NF-κB p65 in primary microglia. L Statistical analysis of the fluorescence intensity in K. Data are presented as the mean ± S.E.M. from at least three dependent experiments. Data in J, L were analysed by two-way ANOVA, and all others were analysed using one-way ANOVA followed by Tukey’s post-hoc test. ns for P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001