Figure 8. Mdm2 promotes the NEDDylation of p53β. One μg p53β was expressed in H1299 cells in the absence (lanes 1 and 5) or presence (lanes 2–3 and 6–7) of 2μg co-expressed Mdm2 or Mdm2 C464 (designated DN, lanes 4 and 8). 5μg of de-NEDDylating enzyme, Nedp1, was co-transfected in lanes 3 and 7. In all cases, 2μg His6-tagged-NEDD8 was co-transfected and DNA concentrations were maintained with empty pcDNA3 control vector. A Ni-agarose purification of His6-tagged NEDDylated species is shown in the right panel and corresponding whole-cell extract in the left panel. β-gal was used as a loading and transfection-efficiency control throughout.