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. 2021 Mar 4;12(3):238. doi: 10.1038/s41419-021-03517-x

Fig. 5. Identification of USP53 binding proteins and effects of FBXO31 on β-catenin degradation through the SCF complex.

Fig. 5

a Instantblue staining of a co-IP mixture separated by SDS-PAGE. The indicated band was extracted for analysis. b hBMSCs were cotransfected with FLAG-β-catenin with either myc-FBXO31 or myc-FBXO31ΔF for 48 h. Transfected cells were incubated with 10 μM MG132 for 6 h, and whole cells were lysed and subjected to IP with anti-myc antibodies. Immunoprecipitates and input protein extracts (Pre-IP) were resolved in SDS-PAGE. c hBMSCs were transfected with pCMV-myc or myc-FBXO31 for 48 h, and whole cell lysates were immunoblotted. d hBMSCs were transfected with pCMV-myc or myc-FBXO31ΔF for 48 h, and whole-cell lysates were immunoblotted. e hBMSCs were transfected with the pCMV-myc or myc-FBXO31 for 48 h. Transfected cells were then incubated with or without 10 μM MG132 for 6 h. Cell lysates were immunoblotted. f hBMSCs were transfected with pCMV-myc, myc-FBXO31, and myc-FBXO31ΔF for 48 h. Cells were harvested and lysed, and whole-cell protein extracts were immunoblotted. g β-Catenin transcriptional activity was measured on day 5 after induction of osteogenesis by TOP/FOP luciferase assays. h HEK293 cells were transfected with the indicated plasmids for 48 h, treated with 10 μM MG132 for 6 h, lysed, subjected to IP with anti-HA antibodies, and immunoblotted. i hBMSCs were transfected with negative control or FBXO31 siRNA for 5 days in osteogenic induction medium. Immunoblot analysis was performed, and data were quantified using ImageJ. j hBMSCs were transfected with empty vector, FBXO31, or GFP-USP53 and then cultured with osteogenic induction medium for 5 days. Immunoblot analysis was performed, and data were quantified using ImageJ. Results are means ± SDs. ns: not significant, **P < 0.01 by one-way ANOVA. n = 3.