Fig. 4: STUB1 promotes GOT2 degradation by ubiquitin.

A–C T24 or UC3 cells transfected with Flag-STUB1 plasmids for 48 h, the cell were analyzed by western blotting and qRT-PCR assay. D HEK293T cells were transfected with Flag-STUB1 plasmids, treated with 50 mg/mL cycloheximide, harvested at different time points, and then immunoblotted with antibodies to GOT2, Flag tag and β-actin. Bottom, quantification of GOT2 protein levels (normalized to β-actin). E Immunoblotting analysis of the ubiquitination of GOT2 in HEK293T cells were co-transfected with Flag-GOT2, HA-ub plasmids cells ectopic expression of Myc-STUB1 or not. Cells were treated with 20 μM MG132 for 6 h before harvesting. F, G Immunoblotting analysis of the ubiquitination of GOT2 in T24 or UC3 cells transfected with or without Myc-STUB1 plasmids. Cells were treated with 20 μM MG132 for 6 h before harvesting. H HEK293T cells were transfected with Myc-STUB1, Flag-GOT2, and HA-ub (WT, K6R mutant, K11 mutant, K27 mutant, K29 mutant, K33 mutant, K48 mutant, and K63 mutant). The analysis was undertaken as described for (E). I Prediction ubiquitin site of GOT2 by alignment of the similarity of GOT2 sequences across multiple species. J Immunoblotting analysis of the ubiquitination of GOT2 in HEK293T cells were co-transfected with Myc-STUB1 plasmids and ectopic expression of GOT2 (wild type, K73A and K396A) or not. K, L HEK293T cells were co-transfected with Myc-STUB1 plasmids, HA-GFP plasmids (internal control) and ectopic expression of GOT2 (wild type, K82A or K364A), treated with 50 mg/mL cycloheximide (CHX), harvested at different time points, and then immunoblotted with antibodies to Flag tag, Myc tag, HA tag and β-actin. Right, quantification of GOT2 protein levels (normalized to β-actin).