Figure 3: UBR4 targets MAT IIα for degradation.

(a) UBR4 overexpression decreases MAT IIα protein. HEK293T cells were transfected as indicated (UBR4 (D) represents a truncated form of UBR4 protein, with substrate-binding and catalytic domains only), followed by MG132 treatment. Cell lysates were directly subjected to western blotting. (b) UBR4 knockdown rescues MAT IIα protein reduced by folate deprivation. Three different siRNA oligos siUBR4-1, -2 and -3 were transfected, respectively, or together (siUBR4-T) into HEK293T cells. Protein levels of MAT IIα were determined by western blotting. UBR4 knockdown efficiency was analysed by qPCR. Values were normalized against β-actin and compared with the relative mRNA of folate (1 mg l^-1) group (set as 1.0). Error bars represent ±s.d. of triplicate experiments. The two-tailed Student’s t-test was used. **P<0.01; ***P<0.001. (c) UBR4 knockdown increases MAT IIα stability. HEK293T cells were transfected with siUBR4 or control. CHX chase experiment was performed and MAT IIα protein was determined by western blotting (left panel). The right panel showcases relative protein amounts of different groups. Error bars represent ±s.d. of triplicate experiments. The two-tailed Student’s t-test was used. **P<0.01; ***P<0.001. (d) UBR4 knockdown blocks folate-deprivation induced-ubiquitylation of MAT IIα. HEK293T cells were transfected as indicated and cultured in folate containing or deprived culture medium. Ubiquitylation assay was conducted. The efficiency of UBR4 knockdown was validated by qPCR. Error bars represent ±s.d. of triplicate experiments. The two-tailed Student’s t-test was used. ***P<0.001.