Figure 3.

Immunoprecipitation with anti-P2X₄ antibody. Antibodies to P2X₄ were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads, were resolved by SDS-PAGE, and Western blots were probed with A) P2X_1, B) P2X₄ or C) Nt5e antibodies. (A) Left and right panels show P2X₁ immunoblots on IP and FT lysates from wild type and P2X₄−/− mice. P2X₁ is highly concentrated in the FT fractions. Minor potential P2X₁ staining appears in the IP lane, however this is due to P2X₄ antibody cross-reacting and pulling down some P2X₁. (B) P2X₄ antibody detects P2X₄ as a band at 70 kDa in wild type IP lane, but is absent in P2X₄−/− mice. The antibody shows minor cross-reactivity to P2X₁ (50 kDa band). (C) An antibody to 5′-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X₄ is ‘clean’ with no non-specific protein binding evident in the IP lanes.