Figure 4

ERG-associated protein with SET domain (ESET) localises to promyelocytic leukaemia (PML) nuclear bodies and is post-translationally modified by small ubiquitin-related modifier (SUMO). (a) ESET (red) localises to distinct, punctate foci in euchromatin regions, as indicated by the absence of DAPI (blue) staining in embryonic stem (ES) cells. Scale bar, 3 μm. (b) ESET (red) localises to distinct, punctate foci that overlaps with PML nuclear bodies (green) in ES cells. Nuclei are labelled in blue. Scale bar, 5 μm. (c) ES cell lysates were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in digitonin-containing buffer and subjected to western blotting (WB) with the antibodies indicated. Rabbit IgG was used as a negative control. (d) Western blot analysis of coimmunoprecipitation experiment in 293T cells transfected with haemagglutinin (HA)-ESET and/or Flag-SUMO-1. S-ESET represents SUMOylated ESET. (e) ES cell lysates were immunoprecipitated (IP) with the indicated antibodies in NP40-containing buffer either in the presence or absence of N-ethylmaleimide (NEM) and subjected to western blotting (WB) using 4% to 15% Tris-HCl gradient gel. A rabbit anti-HA antibody was used as negative control. (f) Quantitative polymerase chain reaction (Q-PCR) analysis of the enrichment of SUMO-1 on different positions along Cdx2 promoter relative to the least enriched region (C5) after normalising against their respective input and IgG controls. Error bars, standard deviation (SD) of three technical replicates.