Expression of P2X3 receptors in the raft domain of trigeminal neurons in situ. A, left, Examples of western immunoblots of trigeminal ganglion lysates from WT or KI mice labeled with an anti-P2X3 antibody. Gel loading is shown with β-tubulin signal. Middle, Histograms compare expression of P2X3 receptors in total lysates from WT and KI ganglia (n = 5). Right, Histograms quantify stronger expression of P2X3 receptors in membrane compartments of KI samples (n = 5, p = 0.0072). Total P2X3 protein level was normalized with respect to β-tubulin. Membrane or cytoplasmic contents were expressed a fraction of total P2X3 expression. B, left, Examples of western blots (48 kDa) of trigeminal ganglion lysates (from WT or KI mice) immunostained with the anti-flotillin antibody. Equal gel loading is shown with β-actin signal. Right, histograms show significant difference between WT and KI samples (n = 5, p = 0.05). C, western blotting of sucrose density gradient fractions of trigeminal ganglion lysates from WT (top rows) or KI (bottom rows) immunostained with anti-P2X3 and anti-flotillin antibodies. Note discrete localization of P2X3 subunits to flotillin enriched fractions. D, left, Example of western blotting of immunopurified P2X3 receptors from membrane raft and non-raft fractions. Total input for P2X3 receptors is also shown together gel loading input with β-tubulin. Flotillin bands indicate the raft fractions. Right, Histograms quantify significantly larger expression of P2X3 receptors in the raft fraction of KI ganglion neurons (p = 0.008; n = 5). The P2X3 expression at cytoplasm level was significantly lower in KI samples vs WT ones (p = 0.05; n = 5).