Fig. 4: USP12 stabilized MDK through deubiquitination.

A MDK-Flag and HA-UB plasmids with USP12, USP12 mutant (C48A) and control vector were co-transfected into HEK293T cells for 36 h. After 6 h of incubation with 10 μM MG132, ubiquitination assay was performed to detect the poly-ubiquitination of MDK. B The endogenous poly-ubiquitination level of MDK in MDA-MB-231 cells after USP12 overexpression was detected by the deubiquitination assay. C Indicated plasmids were transfected into HEK293T cells; 36 h after transfection, the cells were treated with MG132 for 6 h and then subjected to ubiquitination assay. D USP12 was stably overexpressed in MDA-MB-231. Immunoblotting showed the protein levels of MDK and USP12. E USP12 was knocked down in MDA-MB-231 cells. Immunoblotting showed the protein levels of MDK and USP12. F USP12 was overexpressed in MDA-MB-231 cells, and the MDK level in the medium supernatant of MDA-MB-231 cells was detected by immunoblotting. G USP12 was knocked down in MDA-MB-231 cells, and the MDK level in the medium supernatant of MDA-MB-231 cells was detected by immunoblotting. H MDK was co-expressed with USP12, USP12 mutant (C48A) and control vector in HEK293T cells. After 36 h, cells were treated with CHX (50 µg/ml) for the indicated time intervals. The expression of MDK and USP12 was detected (left panel), and the intensity of MDK expression was quantified by ImageJ software (right panel). I USP12 was knocked down with shRNA in MDA-MB-23 cells, and the MDK half-life was analysed by CHX pulse-chase assay with immunoblotting (left panel) and quantified (right panel).