Figure 3.
Interaction of human p53 and protein phosphatase 5 (PP5). (A) Split-ubiquitin interaction assay. Clones expressing p53 (p53-CRU bait construct) and PP5 (Nub-PP5 prey) were spotted in serial dilutions onto medium lacking uracil (-URA), medium containing 5FOA (+FOA), and control plates selective for the presence of plasmids (C). Interaction is indicated by lack of growth on -URA, paralleled by 5-FOA resistance. A clone co-transformed with p53 and empty prey vector (NubI) served as negative control. A previously identified bait construct with an inactive URA3p phenocopying a true interaction event was also included. (B) GST-pull down experiment. Purified Strep-tagged PP5 (Strep-PP5) expressed from E. coli was incubated with GSH-agarose beads loaded with E. coli expressed GST-p53 fusion protein, or GST alone. Corresponding aliquots of Strep-PP5 ‘input’, ‘unbound’ and ‘bound’ fractions were electrophoresed using SDS-PAGE, and electroblotted to nitrocellulose. Detection was performed first by using a Strep-tactin-HRP conjugate, followed by redetection using an α-GST-HRP conjugate. The asterisk indicates the specific signal.