Fig. 5: RNF126 regulates the stability of PTEN.

A The 293T cells were transfected with GFP-PTEN and FLAG-RNF126 plasmids at different doses 36 h, then the cells were collected, and anti-GFP antibodies were detected by western blotting to determine the level of exogenous PTEN. B GFP-PTEN and FLAG-RNF126 were transfected into 293T cells for 24 h, then the cells were treated with dimethyl sulphoxide (DMSO), 10 μM MG132 (#S2619, Selleck) or 20 μM chloroquine (CQ, #S8808, Selleck) for 8 h. The cells were collected and anti-GFP antibodies were detected by western blot. C FLAG-RNF126 plasmid was transfected into 293T cells, and then 100 μg/ml cycloheximide (CHX, #S7418, Selleck) was respectively added at the specified time points for 0 h, 4 h, 8 h, and 12 h. Then, the cells were harvested, and western blot detected GFP antibodies to determine the half-life of GFP-PTEN protein. D The RNF126 siRNA was transfected into BCa 5637 cells for 36 h, and then 100 μg/ml CHX was added at specified time points for 0 h, 4 h, 8 h, 12 h, and collect cells. The half-life of endogenous PTEN protein was determined by western blot. E, F The ImageJ v1.45 software was used to quantified PTEN protein abundance. The relative level of PTEN protein plotted as indicated. G The in vivo ubiquitination assay showed that RNF126 poly-ubiquitinated PTEN. HA-Ub, GFP-PTEN and FLAG-RNF126 plasmids were transfected into 293T cells for 36 h. The cells were treated with 10 μM MG132 or DMSO for 8 h before harvest. H Graphic model of RNF126 affecting proliferation and metastasis through ubiquitination and degradation of PTEN in BCa.