Figure 3: Pirt interacts with P2X3 to inhibit its activity.

The HEK293T cells were transfected as indicated. (a) The co-immunoprecipitation of Pirt and P2X3 in HEK293T cells. (b) The sample traces of inward currents induced by 10 μM α,β-meATP in HEK293T cells. (c) The histogram of P2X3 current density. **P<0.01, two-tailed unpaired Student’s t-test, n=38. (d) The concentration–response curves for α,β-meATP-elicited currents in HEK293T cells. The smooth lines show the values predicted by a sigmoidal fit. n=12 for each concentration. (e) An example of an immunoblot obtained from the biotinylation assays. (f) The total protein level and cell surface expression of P2X3 in HEK293T cells was not altered by Pirt. The data were acquired from three individual experiments. (g) The sample traces of inward currents. Ten micromole α,β-meATP was applied six times with 50-s intervals for each time. (h) The plotting of P2X3 peak currents, n=38. (i) The histogram of P2X3 peak current recovery after 50-s intervals (the peak current induced by the second 10 μM α,β-meATP application/the first 10 μM α,β-meATP application). P>0.05, two-tailed unpaired Student’s t-test, n=38. (j) Co-immunoprecipitation of Pirt and P2X3 in DRG neurons from WT mice. (k) The sample traces of inward currents induced by 30 μM α,β-meATP in neurons from WT mice or Pirt KO (Pirt^−/−) mice. (l) The histogram of the current densities of P2X3. **P<0.01, two-tailed unpaired Student’s t-test, n=10. (m) The concentration–response curves for α,β-meATP-elicited currents in DRG neurons. The smooth lines show the values predicted by a sigmoidal fit; n=12 for each concentration. Error bars represent s.e.m.