Fig. 8.

Immunoreactivity, co-immunoprecipitation and membrane expression of rASIC3 and rP2X3Rs in rat DRG neurons. a ASIC3 and P2X3R immunoreactivities in rat DRG neurons cultured in the presence (upper panel) or absence (lower panel) of nerve growth factor (NGF). Hoechst (Hoe) was used to stain the cell nuclei. b Examples of immunoprecipitation (IP) of DTSSP-treated extracts of primary sensory neurons with P2X3 antibodies revealed in western blots (WB) with anti-ASIC3 antibodies. Incubation of neuronal cultures at pH 6.8 or 7.5 has no effect. ASIC3 signal is not found after immunoprecipitation with unrelated antibodies (IgG). Input ASIC3 and P2X3 contents in total extracts are also shown. β-Actin is used as gel loading control. c Examples of immunoprecipitation of P2X3 receptors (P2X3, upper panel) with ASIC3 channels in control conditions (scramble) and after P2X3 receptor silencing (siP2X3). No P2X3/ASIC3 signal was found after siP2X3 treatment. Western blot with anti-ASIC1 or anti-ASIC2 antibodies gave no signal. Pull down with unrelated antibody (IgG) gave no signal. Quality of input lysates and equal gel loading is shown (lower panel, total lysates). d Surface expression of P2X3Rs. Example of membrane protein biotinylation experiments in CHO cells transfected with plasmids encoding for ASIC3 alone or ASIC3 plus P2X3Rs (upper panel, surface). Quality of total protein extracts and controls for equal gel loading are also shown (bottom panel, total lysates). The scale labels in the right upper and lower panels of (a) were 20 µm