Figure 1

SUMO-1 is conjugated to HDAC4 in vivo. (A) Anti-HDAC4 western blot of HeLa whole-cell extract (WCE) from cells untransfected (ENDO) or transfected with HDAC4 expression plasmid (TFXN). The filled triangle shows the position of the higher molecular weight HDAC4 species. To yield equivalent signals, 20-fold less extract was loaded from transfected than from untransfected cells. (B) HeLa cells were co-transfected with the indicated plasmids (1 g each) and empty expression vector up to 2 g. WCE (lanes 1–5) and NTA precipitates (lanes 6–10) from transfected cells were analysed by western blotting using the anti-HDAC4 rabbit polyclonal antibody. The positions of unmodified and modified HDAC4 are indicated. The open and filled triangles mark the position of conjugates with exogenous His- (or HA-) SUMO-1 and endogenous SUMO, respectively. (C) Immunofluorescence labelling of HeLa cells co-transfected with expression plasmids encoding HDAC4 (labelled with anti-HDAC4 serum and FITC–anti-rabbit secondary antibodies, left panel) and HA-SUMO-2 (detected with anti-HA mAb 12CA5 and Texas Red-anti-mouse secondary antibody, middle panel). Co-localization appears yellow in the merged confocal image (right panel). The arrowhead indicates a SUMO-positive/HDAC4-negative nuclear aggregate probably corresponding to a PML nuclear body.