Figure 6.

HDAC4 C292 is a key SUMO E3 ligase activity site. (A) Domain analysis of HDAC4–IκBα interaction. HEK293T cells (1 × 10⁶) were transiently transfected with the indicated plasmids (3 μg each) for 36 h followed by coIP experiments and immunoblotting analysis with the indicated antibodies. (B) HDAC4 C292 as a SUMO E3 ligase activity site. HEK293T cells (1 × 10⁷) were cotransfected with Cre-SUMO1, Ubc9, Flag-IκBα, and the indicated expression plasmids for WT or mutant HDAC4. Sumoylation assays and immunoblotting analysis were performed 36 h after transfection. (C) HDAC4 C292S mutant weakens the sumoylation of IκBα in in vitro sumoylation assays. The in vitro sumoylation assay was performed as described in Materials and methods. (D) SUMO1 is mainly conjugated to IκBα K21. The experiments were performed as in A. Data are representative of three independent experiments.