SUMO-3 blocks the ubiquitylation of Glis2.

(A) HEK 293T cells were transfected with the plasmids as indicated. After 24 hours of transfection, F.Glis2 was precipitated using FLAG-M2 beads; Glis2 ubiquitin species were detected using anti-HA antibody (Ubi-Glis2, top). (B) HEK 293T cells transfected with Glis2 alone and with SUMO-3 and PIAS4 were treated with cycloheximide (30 μg) to prevent protein synthesis. After 9 hours of treatment, protein turnover of Glis2 was analyzed by Western blotting using a V5 antibody. Anti-β-actin staining confirmed equal protein loading. (C) The graph demonstrates the fraction of Glis2 remaining after 3 to 9 hours of cycloheximide treatment in the presence or absence of SUMO-3/PIAS4.