Fig. 4
Heteromeric association among P2Y receptors. a Immunoprecipitation (IP) of cell lysates visualized by western blot (WB) analyses. a Immunoprecipitated receptors P2Y2 and P2Y4 were visualized with an anti-P2Y1 antibody. P2Y1 receptors immunoprecipitated by the same antibody were used as positive controls. Additionally, immunoprecipitated receptor P2Y1 was seen with either anti-P2Y2 or anti-P2Y4 antibodies. Unspecific immunoprecipitates of rabbit normal serum were used as negative controls (NC). b Immunopreciptation with infrared fluorescent secondary antibody: P2Y1 receptors were immunopreciptated with goat antibodies. After SDS-PAGE the transferred proteins were revealed with rabbit antibodies and analyzed with donkey anti-rabbit antibodies to different fluorophores. First for P2Y1 (red), and after membrane stripping for P2Y2 (green). The whole protein lysate (WP) was used as a control. c Confocal images show the colocalization of P2Y1 receptor with P2Y2 or P2Y4 receptor. FRET signal evidence that these receptors are spatially close. d To corroborate confocal images, emission spectra of samples are shown (Ex. 488). e Analysis of FRET ratios were calculated as explained in the Methods section