Figure 5

Delocalisation of ERG-associated protein with SET domain (ESET) from promyelocytic leukaemia (PML) nuclear bodies promotes the interaction of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4. (a) In PML (green)-depleted embryonic stem (ES) cells (white border), ESET (red) punctate foci were delocalised from PML nuclear bodies. Nuclei are labelled in blue. Scale bar, 3 μm. Cultures also contain cells which were not depleted of PML, where ESET exhibit punctate foci (arrow heads) associated with PML bodies. (b) ES cells transfected with Pml short hairpin RNA (shRNA) for 4 days were immunoprecipitated (IP) with anti-ESET antibody (kind gift of HH Ng; see text) under mild conditions in buffer containing digitonin and ethidium bromide, and were subjected to western blotting (WB) with the antibodies indicated. Note that more Oct4 is precipitated by ESET in Pml knockdown ES cells (*lane 4, top panel). Asterisk (lane 3, middle panel) indicates PML being precipitated by ESET in control cells transfected with an empty vector but not in Pml knockdown ES cells (compare with lane 4, middle panel). (c) ES cells transfected with Pml shRNA for 4 days were immunoprecipitated (IP) with anti-Oct4 antibody in buffer containing NP40, N-ethylmaleimide, and ethidium bromide and subjected to WB using 4% to 15% Tris-HCl gradient gel. More SUMOylated ESET was precipitated by Oct4 in two independent experiments (Exp 1 and Exp 2) in Pml knockdown cells as indicated (*; lane 4 and 6, top panel).