**5. Data analysis: Comparison of the frequencies of monomers, homooligomers, and hetero-oligomers between P2Y1R/A1R and P2Y2R/A1R**

Gold-staining was quantified in the following way. Firstly, gene-transfected HEK293T cells exhibiting the highest number of total immuno-reacted gold particles were defined as 100% labeling. Since co-transfected HEK293T cells that displayed unique pharmacology in our previous study (Suzuki *et al*., 2006) exhibited more than 20% hetero-oligomeric gold particles, we used this number as a threshold in the current study. Thus, cells with more than 20% hetero-oligomeric particles were defined as being "significantly stained", and those with 20% or less were defined as "not significantly stained".

The proportions of relative distributions for A1R and P2YR between cell surfaces and inner cytoplasmic membranes were clearly different (Fig. 6). The tendency for the proportional distribution of A1R and P2YR at the surface of HEK293T cells concur with data from brain

Fig. 6. Comparison of the relative distributions for A1R- and P2Y2R-conjugated gold particles between the cell surface and inner cytoplasmic membranes.

tissues. Based on this, the numbers of immunogold particles at the surface of each cell type in brain tissues were determined. We defined single particles located independently as monomers (A1R and P2YR in Fig. 7), complexes composed of clusters of the same-sized gold particles as "homo-oligomers" (A1R-A1R or P2YR-P2YR in Fig. 7), and those of different sized gold particles as "hetero-oligomers" (A1R-P2YR in Fig. 7). Separate calculations were carried out for particles in Purkinje cells (Fig. 7A, C), hippocampal pyramidal neurons (Fig. 7B, D), and cortical neurons (Fig. 7E). To do this, gold particles were counted the number of in three cells for each region. We previously counted immunogold particles in co-transfected HEK293T cells (Namba *et al*., 2010). The total number of immunoreactive gold particles on

Fig. 7. Bar graphs comparing the relative distributions of A1R(A1)/P2Y1R(Y1) immunoreactive elements in Purkinje cells (A) Hippocampal pyramidal cells (B), and A1R(A1)/P2Y2R(Y2) immunoreactive elements in Purkinje cells (C), Hippocampal pyramidal cells (D) and Cortical neurons (E). P2YR-P2YR, A1R-A1R and A1R-P2YR oligomers are indicated by Y1-Y1 (Y2-Y2), A1-A1 and A1-Y1 (A1-Y2), respectively. The total number of immunoreactive gold particles on the cell surface was defined as 100%. Each column represents the average frequency (± SD) from three cells. Data describing the average numbers of gold particles are shown in tables under the graphs. Data represent the mean of three independent experiments.

each cell surface was defined as 100%. From a total of 12 photos from each brain area (i.e., 36 photos) and from transfected cells that were reacted under the same conditions as the brain sections for each immunostaining, we selected three photos of each specimen containing whole cells for comparison.

We then counted gold particles on the surfaces of cells in the cerebellum, hippocampus and cortical neurons, and classified them as monomers, homo-oligomers, or hetero-oligomers. While the homo-oligomerization ratios (A1R-A1R/P2YR-P2YR) displayed different patterns between Purkinje cells and hippocampal pyramidal cells, the rates of hetero-oligomerization were particularly prominent in hippocampal pyramidal cells among them. Curiously, the frequency of A1R or P2Y1R hetero-oligomerization was slightly higher than that of A1R or P2Y2R in both tissues (Fig. 7A, B). This indicated that the hetero-oligomerization of A1R or P2Y1R are the dominant form in both Purkinje cells and hippocampal pyramidal cells.
