FIGURE 8.

Efficient cell surface expression of A2BR requires A2AR co-expression. A, HA-A2BR and empty vector (Vec), FLAG-A2AR (FA2AR), or FLAG-β2AR (Fβ2AR) were transiently co-transfected to AD-293 cells. The cells were lysed in 1% CHAPS-containing buffer and subjected to immunoprecipitation using rat mAb to HA or rabbit polyclonal antibody to FLAG after 72 h of transfection. The immunoprecipitates were washed and then analyzed by SDS-PAGE and immunoblotting using mouse mAb to HA or FLAG. Not, represents nontransfected cells. 5% of the total cell lysate was subjected to SDS-PAGE and blotted with mAb to β-actin as a loading control. The asterisk indicates IgG heavy chain. B, the cell surface expression of each co-transfectant was determined by enzyme-linked immunoassay. Relative surface expression of HA-A2BR were adjusted by total expression levels and expressed as percent means ± S.D. of HA-A2BR plus empty vector-transfected cells (n = 6). Shown is a representative of three independent experiments. C, HA-A2AR and Myc-A2BR were transiently co-transfected to AD-293 cells. The cells were lysed in 1% CHAPS-containing buffer and subjected to immunoprecipitation (IP) using rat mAb to HA (A2AR) or rabbit polyclonal antibody to Myc (A2BR) after 72 h of transfection. The immunoprecipitates were washed and then analyzed by SDS-PAGE and immunoblotting (Wb) using mouse mAb to HA (A2AR) or Myc (A2BR). D, immunofluorescence localization of HA-A2AR and Myc-A2BR in stably co-transfected AD-293 cells. Cells were immunostained with rat mAb to HA and mouse mAb to Myc. The bound primary antibodies were detected by using either Alexa Fluor 488 (green)- or Alexa Fluor 594 (red)-conjugated goat anti-rat or mouse IgG antibody. Cells were analyzed by immunofluorescence with confocal microscopy. Merged images are shown in yellow. White bar, 10 μm.