Fig. 4: Usp26 deficiency facilitates osteoclastic differentiation of BMMs by decreasing IκBα.

A Volcano plots of differentially expressed genes in Usp26^−/− osteoclasts (Usp26 KO) as compared with WT controls (WT). The blue and red dots represent the down- and upregulated genes, respectively. B KEGG enrichment analysis of the correlated signal pathway to the differently expressed mRNAs between Usp26^−/− and WT osteoclasts. C Western blot analysis of P-p65 in BMMs obtained from Usp26^−/− mice and littermate controls with or without osteoclastic differentiation. D, E NF-κB transcriptional activity in BMMs induced by osteoclastic differentiation with or without different doses of Usp26 overexpression. F Representative of P-p65 IHC staining in femurs of 5-month-old Usp26^−/− mice and their WT littermates (n = 5). Red and black bars represent 25 and 100 μm, respectively. G Western blot analysis of IKKα, IKKβ, IKKγ, and IκBα expression in mouse BMMs. H Co-immunoprecipitation of USP26 with endogenous IKKα, IKKβ, IKKγ, and IκBα in mouse BMMs. I Representative of IκBα IHC staining in femurs of 5-month-old Usp26^−/− mice and their WT littermates (n = 5). Red and black bars represent 25 and 100 μm, respectively. J Western blot analysis of IκBα expression in BMMs isolated from Usp26^−/− mice and their WT littermates with or without osteoclastic differentiation. K Western blot analysis of IκBα and P-p65 expression in Usp26^−/− BMMs with or without Nfkbia overexpression. L, M The osteoclastic genes expression and TRAP staining of Usp26^−/− osteoclasts with or without Nfkbia overexpression. N The schematic graph reflects that decreased IκBα results in increased NF-κB activation and enhanced osteoclastic differentiation in Usp26^−/− BMMs. O, P Co-immunoprecipitation of USP26 with ectopically expressed Flag-tagged IκBα in 293 T cells. Q Overexpression of USP26 decreases the level of ubiquitinated IκBα. R Western blot analysis of ectopically expressed IκBα protein in 293 T cells with or without USP26 overexpression and treated with cycloheximide (CHX) for indicated time intervals. S The schematic graph reflects the underlying mechanisms of USP26 in decreasing IκBα degradation by reducing the level of ubiquitinated IκBα in 293 T cells. **P < 0.01, ***P < 0.001. P values were analyzed by one-way ANOVA. All data are representative of two to three independent experiments.