Figure 6. Immunohistochemistry and Western blotting of P2Y₁ purinoceptors.

A, pacemaker cell fixed and stained with P2Y₁ antibody and fluorescent secondary antibody. Bar indicates 6 μm. B, Western blots demonstrating P2Y₁ expression in toad tissues. Total protein extracts were subject to 10 % SDS-PAGE and probed with P2Y₁ antiserum in the absence (lanes 2–4) and presence (lanes 5–7) of epitope peptide. One band of about 57 kDa was observed in the rat aorta sample that is known to express P2Y₁ receptors. This band is probably the glycosylated form of P2Y₁. The same band was present in the toad samples, whereas an additional band of about 114 kDa was also seen in these samples. This band may be a dimer of the P2Y₁ receptor. Preincubation with the epitope peptide greatly attenuated the 114 kDa band and abolished the 57 kDa band, suggesting specific labelling.