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.