mGluR-interacting domains on PrP^C.

<p><b>(A)</b> Brain homogenate from wild-type (C57BL/6J) and <i>Prnp</i>^<i>o/o</i> mice was subjected to immunoprecipitation by POM1 followed by immunoblotting using polyclonal anti-mGluR5 (right) or anti-mGluR1 (left) and anti-PrP^C antibodies. Control conditions (POM1 blocked by recombinant PrP^C) were run in parallel. The typical mGluR bands of 250kDa and 150kDa were detected in wild-type extract only when immunoblotting with mGluR1 or mGluR5 antibody. Total brain extracts were in parallel subjected to Western blot analysis to control for endogenous levels of mGluR5/1 and PrP^C. <b>(B-C)</b> Mapping the mGluR5 and mGluR1 interacting domains on PrP^C. Brain homogenate from <i>Tga20</i>, <i>Prnp</i>^o/o (ZH3) and amino proximal deletion mutants of PrP^C was subjected to immunoprecipitation by anti-mGluR5 <b>(B)</b> or anti-mGluR1 <b>(C)</b> antibodies. For detection, we used polyclonal antibodies to mGluR5, mGluR1, and PrP^C. Deletions encompassing residues 111–134 of PrP^C reduced its interaction with mGluR5, whereas deletions of residues 51–90 or 11–134 decreased the interaction with mGluR1. Total brain extracts (TEs) were subjected to Western blot analysis to control for endogenous levels of mGluR5/1 and PrP^C. Densitometric quantitation of the PrP^C signal was normalized over the ratio of PrP/Actin signal in TEs. N = 3–5; One-way ANOVA followed by Tukey’s post-hoc test. Asterisk: P<0.05.