Fig. 4: Effects siKDM2A and daminozide on the K48-Ub of nuclear β-catenin after tGCI with or without hypoxia.
A Immunoprecipitation assay showing the K48-Ub of nuclear β-catenin in CA1 of tGCI and HPC groups. Data are expressed as percentage of value of sham-operated animals. *p < 0.05 versus Sham animals (p = 0.006 in the tGCI group, p = 0.698 in the HPC group, One-Way ANOVA Test, p = 0.041 at tGCI 26 h, p = 0.050 at tGCI 50 h, Tamhane post-test), #p < 0.05 versus tGCI group at the same time point (p = 0.007 at 26 h, unpaired t-test, p = 0.015 at 50 h, Mann-Whitney test, n = 6 in each group). B Representative images of western blot showing the expressions of KDM2A, CUL1 and SKP1 in nuclear fraction after tGCI with or without siKDM2A administration. Lamin B1 is used as loading controls to normalize the relative expressions of nuclear proteins of interest. Data are expressed as percentage of Sham animals value. *p < 0.05 versus Sham group with NC (p < 0.001 for KDM2A, n = 4; p = 0.045 for CUL1, n = 8; p = 0.015 for SKP1, n = 6, unpaired t-test), &p < 0.05 versus the same group with NC (For KDM2A, p < 0.001 in the Sham and tGCI group with siKDM2A; For CUL1, p = 0.363 in the Sham group with siKDM2A, p = 0.218 at the tGCI group with siKDM2A; For SKP1, p = 0.097 in the Sham group with siKDM2A, p = 0.001 in the tGCI group with siKDM2A; unpaired t-test). C Immunoprecipitation assays showing the effects of siKDM2A administration on the K48-Ub of nuclear β-catenin and the interaction between β-catenin and three subunits of SCFKDM2A within nucleus of Sham and tGCI rats. Data are expressed as percentage of Sham animals value. *p < 0.05 versus Sham group with NC (p = 0.046 for K48-Ub/β-catenin, n = 7; p < 0.001 for KDM2A/β-catenin, n = 5; p = 0.028 for CUL1/β-catenin, n = 5; p < 0.001 for SKP1/β-catenin, n = 6; unpaired t-test, &p < .05 versus the same group with NC (for K48-Ub/β-catenin, p = 0.537 in the Sham group with siKDM2A, p = 0.006 in the tGCI group with siKDM2A; for KDM2A/β-catenin, p = 0.617 in the Sham group with siKDM2A, p = 0.001 in the tGCI group with siKDM2A; for CUL1/β-catenin, p = 0.233 in the Sham group with siKDM2A, p = 0.006 in the tGCI group with siKDM2A; for SKP1/β-catenin, p = 0.706 in the Sham group with siKDM2A, p = 0.022 in the tGCI group with siKDM2A; unpaired t-test). D Representative images of western blot showing the expression of nuclear β-catenin in CA1 of Sham and tGCI groups with or without daminozide administration. Lamin B1 is used as loading controls to normalize the relative expressions of nuclear proteins of interest. Data are expressed as percentage of Sham animals value. *p < 0.05 versus Sham group with vehicle (p = 0.004, unpaired t-test), &p < 0.05 versus the same group with vehicle (p = 0.118 in the Sham group with daminozide, p = 0.045 in the tGCI group with daminozide, unpaired t-test, n = 4 in each group). The nuclear fraction purity was assessed by absence of GAPDH staining in Figure B and D. E Immunoprecipitation assays showing the level of nuclear K48-ubiquitinated β-catenin and me-β-catenin in CA1 of Sham and tGCI groups with or without daminozide administration. Methylation was detected by western blot using antimethyl-lysine antibody. *p < 0.05 versus Sham group with vehicle (p = 0.019 for K48-Ub/β-catenin, n = 4; p = 0.022 for Methyl-K/β-catenin, n = 7, unpaired t-test), &р < 0.05 versus the same group with vehicle, injection intracerebroventricularly with ddH2O (for K48-Ub/β-catenin, p = 0.294 in the Sham group with daminozide, p = 0.023 in the tGCI group with daminozide; for Methyl-K/β-catenin, p = 0.252 in the Sham group with daminozide, p = 0.045 in the tGCI group with daminozide, unpaired t-test). Each bar represents the mean ± SD (error bars).
