Supplementary Figure 6: Control studies for biotinylation experiments.

To measure cell surface expression of mGluR1 and mGluR5, biotinylation experiments were performed as described in the Methods section in the main text. Briefly, NAc tissue was dissected and incubated with a membrane impermeant biotinylating reagent (1 mM sulfo-NHS-S-S biotin) to selectively modify surface-expressed proteins. Biotinylated proteins bound to NeutrAvidin beads (Bound fraction) were then separated from the non-biotinylated proteins (Unbound fraction) by centrifugation. To assess distribution of group I mGluR dimers in NAc tissue, equal amounts (15 μg) of the starting material (Input), the Bound fraction, and the Unbound fraction were analyzed by SDS-PAGE and immunoblotting for mGluR1 or mGluR5. (a) The majority of mGluR1 or mGluR5 dimer was recovered in the Bound fraction, consistent with evidence that the dimer represents the functional surface-expressed receptor pool (e.g., Jingami et al., 2003). This observation was reproduced over ten times. (b) Additional controls were performed to verify that intracellular proteins such as tyrosine hydroxylase (TH) are not detected in the Bound fraction. This observation was reproduced twice.