Figures & data
TABLE 1. Gene identities obtained from yeast two-hybrid screening
Figure 1. PrPC interacts with 10 different ligands in cross-mating assay. Bait strain expressing PrPC and the Lac-Z reporter gene were mated with target strains expressing the putative PrPC interactors. X-gal was used to score positive interactions (blue color development). pBait and pTarget were used as a positive control for interaction, since they express known interaction partners. The empty bait vector (pGilda), which expresses only Lex A domain, was used as negative control.
TABLE 2. Ten putative PrPC ligands
Figure 2. Stub1 is expressed in both olfactory epithelium (OE) and brain. (a) Protein extracts prepared from mouse brain and OE were analyzed by western blotting with anti-Stub1 (upper panel) antibodies. Anti-β-actin was used as loading control (lower panel). (b) Protein relative values were quantified using ImageJ Software® and normalized to β-actin. The results are representative of 4 independent experiments. Statistical analysis was conducted with GraphPad Prism (values were expressed as mean ±SE. Student's t test was used for statistical analysis and P<0.05 was considered significant).
Figure 3. PrPC interacts with Stub1. (a) A pull-down assay was performed using mouse brain extract incubated with His6-PrPC bound to Ni-NTA-agarose beads (+) or Ni-NTA-agarose alone (−). Pulled-down proteins were analyzed by western blotting (WB) employing anti-Stub1 antibody (upper panel). The same membranes subjected to pull-down assay were re-probed with anti-his-tag antibody to attest that recombinant PrPC was recovered from the beads (lower panel). HEK293T cells protein extracts overexpressing GFP-PrPC and Myc-Stub1 (b) or olfactory epithelium (OE) extracts (c) were immunoprecipitated with anti-PrPC or pre-immune serum (PI). Co-precipitated proteins were analyzed using an anti-Stub1 antibody (upper panels). The same membranes were re-probed with anti-PrPC (lower panels) to confirm that PrPC was precipitated during IP-reaction.
