A) Schematics of the PML protein depicting its functional domain. RING indicates the RING finger domain while B1 and B2 represent the B-boxes. S indicates SUMO protein modification of the lysine residues at the indicated positions (boxed in red). B) Chromatographs depicting successful mutations of the lysine sites to arginine in the PML protein through PCR mutagenesis. Chromatograms of (i) PML K65R, (ii) K160R, (iii) K490R, (iv) K65/160R, (v) K65/490R, (vi) K160/490R and (vii) K65/160/490R plasmids. The boxed codons indicate the location of the successful mutations. C) Western blot analysis showed that PML K160/490R double and K65/160/490R triple mutants are not modified by SUMO. JFCF-6/T.1R cells were transfected with the indicated plasmids and cells were harvested 48 h after transfection in the presence and absence of NEM. The use of NEM preserves SUMO-conjugated proteins. The SUMO protein adds about 10–20 KDa in molecular weight to that of the protein in its native form. The slower migrating band is indicative of SUMO-modified PML. All PML constructs are HA-tagged and the blots were probed with the indicated antibodies (anti-HA: 1:5000, anti-actin: 1:2000). Actin was used as a loading control.