Figure 2. PrP^C Interacts with mGluR5.

A Aβo (250 nM) binding to COS7 cells expressing mGluR5, PrP^C, or co-expressing mGluR5 with PrP^C (upper). Protein expression was confirmed by immunofluorescence (bottom). Scale bar, 50 μm.

B Dose-response for Aβo binding to COS7 cells from experiments as in A. Mean±sem, n=3 experiments.

C HEK293T cells were transfected with vector for either mGluR5 or PrP^C, or co-transfected for mGluR5 and PrP^C. After treatment with 0 or 1 μM Aβo for 1 h, lysates (input) and anti-PrP^C immunoprecipitates were immunoblotted with either anti-mGluR5 or anti-PrP^C.

D HEK293T cells were transfected with vector for PrP^C or co-transfected with different Myc-tagged mGluRs and PrP^C, as indicated. Lysates (input) and anti-Myc immunoprecipitates were immunoblotted with either anti-Myc or anti-PrP^C antibodies.

E Indicated fractions (20 μg protein) were prepared and analyzed by immunoblot with anti-PrP^C, anti-mGluR5, anti-EphB2, anti-PSD95, anti-synaptophysin, and anti-actin antibodies.

F Brain lysates from WT or Prnp −/− mice were cross-linked with the cleavable DTSSP. Whole lysates (5% Input) and anti-PrP^C immunoprecipitates were immunoblotted with anti-mGluR5, anti-mGluR8, anti-NR2B, anti-GluR1 or anti-PrP antibodies. Asterisk, Ig light chain.

G His-tagged human PrP^C was incubated with DIV21 WT or Grm5 −/− neurons for 1 h. Neurons were then fixed and stained with human-specific anti-PrP^C antibody and Alexa-568 secondary antibody with rhodamine-phalloidin as counterstain. Scale bar, 10 μm.

H Quantification of His-PrP^C bound to WT or Grm5 −/− neurons. His-PrP^C immunofluorescence was measured by ImageJ in segments of primary dendrites 10 μm from the soma and background-subtracted. Mean±sem, n=3 embryos for each genotype.***, P<0.001; Student’s two-tailed t test.

See also Fig. S2.