Effect of MβCD treatment on WT or KI ganglion cultures. A, Left, cholesterol staining (with filipin) of WT or KI trigeminal neurons (arrow) before and after MβCD (30 min, 10 mM). Satellite cells (arrowhead) are also stained. Bar=10 μm. Right, fall in cholesterol staining after MβCD (indicated by +) for WT or KI neurons (n= 55); p=0.02 for WT before and after MβCD; p=0.03 for untreated WT vs untreated KI; p=0.01 for KI before and after MβCD. B, CTX-B staining of WT and KI trigeminal neurons before and after MβCD. Bar=10 μm. Right, Large reduction in CTX-B staining after MβCD in WT (p<0.001) and in KI cells (p<0.001). The CTX-B staining of untreated KI cells was significantly larger vs untreated WT neurons (p<0.002); n=62 cells. C, Raft (R) and non rafts (NR) fractions from WT and KI lysates before or after MβCD. Non raft fractions were cytosolic samples collected from supernatants after ultracentrifugation with a swing bucket rotor. Conversely, membrane pellets labelled with flotillin were considered to be raft fractions. Immunolabelling was performed with anti-P2X3 receptor and anti-flotillin antibodies. The histograms (right) quantify the distribution of P2X3 receptors to raft and non- raft compartments of WT or KI cultures: the raft fractions of KI was significantly larger than the corresponding WT ones (*: p=0.004, n=3). After 30 min application of MβCD there was redistribution of P2X3 receptors to cytosolic fractions of WT or KI samples. ** indicates p=0.0038, while *** indicates p=0.001 (n=3). D, example of immunoblotting of P2X3 receptors from supernatants and pellets collected after ultracentrifugation with a fixed angle rotor. The flotillin labelled membrane fractions of KI cultures were largely decreased by overnight pretreatment with ω-agatoxin (300 nM; selective blocker of P/Q channels) as indicated by the histograms (right; * shows p=0.02, n=4).