Fig. 3: FBXO7 protein mediates the degradative ubiquitination of INF2 protein.

A Representative images of IHC analysis of FBXO7 and INF2 proteins expression on ECa and normal endometrium tissues (142 samples, including 25 normal tissues and 117 ECa tissues). Scale bar, 100 μm. ***p < 0.001 vs. the Normal group. B Staining patterns of IHC analysis based on (A). ***p < 0.001 vs. the Normal group. C The score of FBXO7 (Left) and INF2 (Right) proteins expression levels based on (A). ***p < 0.001 vs. the Normal group. D Correlation analysis of the protein expression levels based on (A and C). r = –0.3889, p < 0.001. E Western blotting of WCLs of HEK-293T cells transfected with FLAG-INF2 plasmid and the increasing Myc-FBXO7 plasmid. All quantitation were normalized to the protein level of endogenous control GAPDH. F Western blotting of WCLs of HEK-293T cells transfected with transfected with FLAG-INF2 plasmid and the increasing Myc-FBXO7 plasmid, and treated with MG132 (20 μM), Bortezomib (200 nM), Chloroquine (100 mM) or DMSO for 8 h before harvesting. All quantitation were normalized to the protein level of endogenous control GAPDH. G Western blotting of WCLs of AN3 CA cells transfected with pCDH-CD513B-FBXO7 plasmid and treated with MG132 (20 μM) or DMSO for 8 h before harvesting. All quantitation were normalized to the protein level of endogenous control GAPDH. H FBXO7 knockout was performed in AN3 CA and HEC-1-A cell lines. Western blotting of WCLs of AN3 CA (Left) and HEC-1-A (Right) cells with FBXO7 knockout and parental. All quantitation were normalized to the protein level of endogenous parental GAPDH. I Western blotting of the products of in vivo ubiquitination assays performed using WCLs and co-IP samples of anti-INF2 antibody from AN3 CA cells transfected with HA-Ub and Myc-FBXO7 plasmids or not, and treated with 20 μM MG132 for 8 h before harvesting to show the ubiquitination of endogenous INF2 protein mediated by FBXO7 protein. J Western blotting of the products of in vivo ubiquitination assays performed using WCLs and co-IP samples of anti-INF2 antibody from HEK-293T cells transfected with Myc-FBXO7 plasmid or not, and treated with 20 μM MG132 for 8 h before harvesting to show the ubiquitin linkage specificity of endogenous INF2 protein mediated by FBXO7 protein. K The half-life of INF2 protein was detected by western blotting of WCLs of AN3 CA cells with FBXO7 overexpression, FBXO7 knockout and parental treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points. All quantitation were normalized to the protein level of endogenous parental GAPDH (Left). Statistics of INF2 protein half-life (Right). Data are shown as means ± SD (n = 5). Experiments in (E, F, G, H, I, J) were repeated three times.