Figure 2

Real time RT-PCR and western immunoblot of ganglia and cultures. A, real time RT-PCR of ganglia and cultures from rat and mouse. Ordinate: relative increment with respect to the ganglion products. For each receptor, amplification values were normalized with β-tubulinIII mRNA levels and compared with the ganglion mRNA levels. GAPDH amplification control was the same in all reactions. B, western immunoblots of equal amounts of neuronal protein lysates (β-tubulinIII) derived from ganglia or culture. Immunodetection of P2X₃, P2X₂ and TRPV1 mature proteins revealed proper migration (a) in accordance with Vulchanova et al.[53] for the predominant P2X₃ form (57 kDa), with Newbolt et al. [54] for the P2X₂ mature receptor (65 kDa) and with Kedei et al. [55] for the TRPV1 mature receptor (120 kDa). Panel B b shows P2X₃ native (45 kDa) and intermediate polypeptides (up to 50 kDa) detected one d in culture (1) but not in the ganglion (T) as reported by Nicke et al. [56] C, relative optical density values of mature receptors at 1–4 days in culture (normalized with respect to β-tubulinIII) and compared to ganglion values. For all experiments shown in A-C n = 3 animals for ganglion or day in culture datapoint (each point is mean ± SEM). *: P < 0.05; **: P < 0.01.