Fig. 1: KDM2A assembles the SCFKDM2A E3 ligase complex and targets nuclear β-catenin for ubiquitylation through the F-box domain.
A Western blot assay showing the expressions of total, nuclear and cytoplasmic β-catenin at 2, 6, 12, 24 h after OGD/R in in primary hippocampal neurons, respectively. GAPDH is used as loading control to normalize the relative expression of whole cellular/cytoplasmic β-catenin and Lamin B1 is used as loading control to normalize the relative expression of nuclear β-catenin. Data are expressed as percentage of value of the control group. *p < 0.05 versus control group (p = 0.232 for total β-catenin, n = 3-5, Kruskal-Wallis Test; p < 0.001 for nuclear β-catenin, n = 6, One-Way ANOVA Test, p = 0.048 at OGD/R 2 h, p = 0.001 at OGD/R 6 h, p = 0.011 at OGD/R 12 h, p = 0.019 at OGD/R 24 h, Tamhane post-test; p = 0.345 for cytoplasmic β-catenin, n = 4, One-Way ANOVA Test). B Effects of MG132 on nuclear and cytoplasmic β-catenin proteins in primary hippocampal neurons at 24 h after OGD/R using western blot analysis. GAPDH and Lamin B1 are used as loading control to normalize the relative expression of cytoplasmic and nuclear β-catenin, respectively. Data are expressed as percentage of value of the control group with DMSO. *p < 0.05 versus the control (ctrl) group with DMSO (p = 0.04 for nuclear β-catenin, p = 0.977 for cytoplasmic β-catenin, unpaired t-test), #p < 0.05 versus the same group with DMSO (p < 0.001for nuclear β-catenin in the ctrl group or OGD/R group, p = 0.211 for cytoplasmic β-catenin in the ctrl group, p = 0.03 for cytoplasmic β-catenin at the OGD/R group, unpaired t-test, n = 3 in each group). C Immunoprecipitation assay showing the K48-Ub of nuclear β-catenin. β-catenin was immunoprecipitated (IP) using anti-β-catenin antibody. IgG antibody was used as a negative control. K48-Ub was detected by western blot using anti-ubiquitin (linkage-specific K48) antibody. Data are expressed as percentage of value of the control group with MG132. *p < 0.05 versus the control group with MG132 (p = 0.018, n = 3-4, unpaired t-test). D Immunofluorescence assay showing that the effects of Flag-KDM2A (a-d, e-h) or Flag-KDM2A-∆F-box (i-l, m-p) plasmids treatment on the fluorescence intensity of β-catenin with or without MG132 treatment. Representative photomicrographs with fluorescent staining of β-catenin (white), Flag (red) and DAPI (blue) in SH-SY5Y cells. White arrows indicate the positive cells expressed exogenous KDM2A protein. Scale bar: 10 μm. E Western blot assay showing the expression of Flag, KDM2A and β-catenin in nuclear lysates of HEK-293T and SH-SY5Y cells which were transfected with the indicated plasmids and then treated with 20 μM MG132 or DMSO for 6 h. The histogram presents the quantitative analyses of nuclear β-catenin levels. PCNA is used as a loading control to normalize the relative expression of nuclear β-catenin. Data are expressed as percentage of value of control group with DMSO. *p < 0.05 versus Flag-Con group with DMSO (p = 0.026 for HEK-293T, p = 0.026 in the Flag-KDM2A group with DMSO, p = 0.207 in the Flag-KDM2A-∆F-box group with DMSO; p = 0.008 for SH-SY5Y, p = 0.01 in the Flag-KDM2A group with DMSO, p = 0.05 at the Flag-KDM2A-∆F-box group with DMSO, One-Way ANOVA Test followed by Bonferroni post-test), #p < 0.05 versus the same group with DMSO (for HEK293T, p = 0.003 in the Flag-Con group, p = 0.005 in the Flag-KDM2A group, p < 0.001 in the Flag-KDM2A-∆F-box group; for SH-SY5Y, p = 0.036 in the Flag-Con group, p = 0.010 in the Flag-KDM2A group, p = 0.002 in the Flag-KDM2A-∆F-box group, unpaired t-test, n = 8 in each group). F Effects of Flag-KDM2A or Flag-KDM2A-∆F-box plasmids treatment on the ubiquitination of Myc-β-catenin were assessed by in vivo ubiquitination assay. HA-Ub and Myc-β-catenin were co-transfected into HEK-293T cells with KDM2A constructs or empty vector control. G Western blots and Immunoprecipitation blots showing the expression of SKP1, CUL1 and Flag and the assembly of SCFKDM2A ligase complex in nuclear lysates of HEK-293T cells which were transfected with the indicated plasmids. HNs, hippocampal neurons; Flag, flag-tagged protein. The nuclear fraction purity was assessed by absence of β-tubulin staining in Figure E–G. Each bar represents the mean ± SD (error bars).
