Figure 4

PML/HSP70 co-aggregation is dependent upon the presence of the pac resistance gene sequence in puromycin vectors. U2OS cells on glass coverslips were transiently transfected with empty vectors and immunostained for HSP70 (green) and PML (red) 48 h after transfection. PML antibodies were detected using a rhodamine-x conjugated goat anti mouse IgG or Cy5 goat anti mouse IgG/pseudocolored red (pBABE hcRed transfected cells only). HSP70 antibodies were detected using Alexa 488 goat anti rabbit IgG or Cy5 goat anti rabbit IgG/pseudocolored green (pBABE GFP transfected cells only). Cell nuclei were counterstained with DAPI (blue). Images were captured at 100× magnification. (A) Representative images of cells transfected with multiple pBABE vectors carrying alternative selection markers demonstrating absence of PML/HSP70 aggregation. (B) Representative images of cells transfected with pBABE Puro del demonstrating absence of PML/HSP70 aggregation. pBABE Puro del was generated by removal of the pac gene sequence from parental vector pBABE PURO.