**2.1 Results: Co-localization of A1R and P2Y1R (P2Y2) in transfected HEK293T cells**

Confocal imaging for studying the GPCRs using transfected HEK293T cells is the most common method of co-localization of GPCRs. In our laboratory, the co-localization of A1R and P2Y1R (P2Y2R) in co-transfected HEK293T cells has been examined by the double immunostaining of HA-A1R and Myc-P2Y1R (or HA-A1R and Myc-P2Y2R) in order to compare localization pattern. Confocal images of co-transfected HEK293T cells double labeled for HA-A1R (red) and Myc-P2Y2R (green) are shown in Fig.1. As co-localization occurred upon the plasma membrane, this data supports the heteromeric association of A1R and P2Y2R. A similar pattern of co-localization for A1R and P2Y2R has been demonstrated in rat brain sections as shown in Fig.2.

Fig. 1. Co-localization of A1R and P2Y2R. *A-C.* Confocal images of double immunostained HA-A1R (A; red), Myc-P2Y2R (B; green), and their merged images (C; yellow) in cotransfected HEK293T cells. The co-localization of HA-A1R and Myc-P2Y2R is evident at the cell surface membrane (C; small arrow). White bar = 50 µm (A-C). Confocal images of double immunofluorescence for HA-A1R (D; red), Myc-P2Y2R (E; green), and their merged images (F; yellow) in co-transfected HEK293T cells are also obtained. Co-localizations of A1R and P2Y1R (F) was detected upon the cell surface membrane, but was not as evident upon inner cellular membranes (F; arrow). Cyan bar = 10 µm (D-F). Fluorescent images were obtained via confocal laser scanning microscopy (Zeiss LSM410, Carl Zeiss, Oberkochen, Germany) at two levels: 30-µm(A-C), and 15-µm(D-F). At each level, serial images were collected at 1-µm intervals through a total sectional thickness of 40-µm. Serial optical sections were recorded using an air objective lens of (20 X and 40X, numerical aperture; 0.6).

Both receptors were localized predominantly upon the cell surface and cytosolic membranes (Fig. 1. A,B). Merged images showed co-localization mainly in cell membranes (Fig. 1. C.). Our negative controls showed no positive signals in non-transfected HEK293T cells, indicating that the immunoreactivity observed in Fig. 1 was specific to the expressed receptors (data not shown).
