Figure 5.

RNF11 suppresses SMURF2 polyubiquitylation in a compartment-specific manner. A, RNF11 antagonizes SMURF2 ubiquitylation in vivo. N-terminally FLAG-tagged wild-type and C716A mutant SMURF2 were co-transfected as indicated with His-tagged ubiquitin and with untagged wild-type RNF11 and a panel of mutants for 48 h in HEK93T cells. Cell lysates were prepared under denaturing conditions and subjected to nickel-affinity agarose purification. Ubiquitylation of SMURF2 and RNF11 was detected by immunoblotting with anti-FLAG or RNF11 antibody as indicated. Transfection with His-ubiquitin alone (lane 1) was included as a negative control. B, subcellular localization of K-less and UIM mutants of RNF11. HeLa cells were transfected with the indicated C-terminal FLAG fusion mammalian expression vectors, fixed after 48 h, and visualized with FLAG antibody and Golgi marker GM130. Scale bar, 10 μm.