Figure 5.

Methylation of RACO-1 is required for protein stabilisation. (A) Overexpression of PRMT1 stabilises RACO-1 expression levels and (B) increases protein half-life as determined by cycloheximide (CHX) chase. (C) Mutation of R98 or R109 to lysine reduces protein expression and this can be rescued by proteasome inhibitor treatment (MG132). (D) Overexpression of PRMT1 increases endogenous RACO-1 expression in BT474 and H727 cells, while (E) silencing of PRMT1 decreases endogenous RACO-1 levels in H727 cells. (F) Ubiquitylation of RACO-1 by MEK1-R4F requires R98 and R109. Cells were transfected as indicated and ubiquitylated RACO-1 resolved by Ni²⁺-NTA affinity purification and immunoblotting with Flag antibodies. (G) Silencing of PRMT1 reduces MEK1-R4F induced RACO-1 ubiquitylation. BT474 stable cell lines expressing scrambled or shPRMT1 silencing constructs were generated (left panel) and then transfected with GFP–RACO-1 and MEK1-R4F. Ubiquitylated RACO-1 was resolved by Ni²⁺-NTA affinity purification and immunoblotting with GFP antibodies (right panel). (H) Overexpression of PRMT1 induces K63-linked ubiquitylation. HEK293T cells were transfected as indicated and ubiquitylated RACO-1 resolved by Ni²⁺-NTA affinity purification and immunoblotting with GFP antibodies.

Source data for this figure is available on the online supplementary information page.

Source Data for Figure 5 ^{(9.4MB, pdf)}