Figure 3

PML aggregates colocalize with HSP70, SUMO-1 and 20S proteasomes in regions distinct from nucleoli in pBABE Puro transfected cells. U2OS cells on glass coverslips were transiently transfected with pCMV Neo (Control) or pBABE Puro empty vector and immunostained 48 h after transfection. Cell nuclei were counterstained with DAPI (blue). Images were captured at 63× (A-C) or 100× (D) magnification. (A) Representative images of cells immunostained for HSP70 (green) and PML (red). PML and HSP70 antibodies were detected using a rhodamine-x conjugated goat anti mouse IgG and Alexa 488 goat anti rabbit IgG respectively. Lower panel displays magnified nuclei (2×) from pBABE Puro transfected cells (panel above) in order to display speckled nuclear staining pattern of HSP70 in transfected cells. (B) Representative images of cells immunostained for SUMO-1 (green) and PML (red). PML and SUMO-1 antibodies were detected using a rhodamine-x conjugated goat anti mouse IgG and Alexa 488 goat anti rabbit IgG respectively. (C) Representative images of cells immunostained for 20S Proteasomes (red) and PML (green). 20S Proteasomes and PML antibodies were detected using a rhodamine-x conjugated goat anti mouse IgG and Alexa 488 goat anti rabbit IgG respectively. (D) Representative images of cells immunostained for Fibrillarin (green) and PML (red). PML and HSP70 antibodies were detected using a rhodamine-x conjugated goat anti mouse IgG and Alexa 488 goat anti rabbit IgG respectively.