Fig. 2. Tyrosine phosphorylation of P2X₇ receptor. (A) Membrane extracts from untransfected (lane 1, negative control) or P2X₇ receptor-expressing HEK293 cells were immunoprecipitated with ecto-P2X₇ Ab, and phosphotyrosine incorporation into the P2X₇ protein was detected by probing with anti-phosphotyrosine Ab PY20. The blot was then stripped and re-probed with C-terminal anti-P2X₇ Ab to confirm that the phosphotyrosine bands (lanes 3, 4 and 6) were the P2X₇ receptor. No tyrosine phosphorylation was detected in control P2X₇-expressing cells (lanes 2 and 5) but after 10 min incubation with the tyrosine phosphatase inhibitor bpV (100 µM) phosphotyrosine was clearly detected (lanes 3 and 6). The level was much reduced if the P2X₇ receptor was activated for 10 min with BzATP (100 µM) prior to incubation with bpV (lane 4). (B) Similar experiments using phosphatase inhibitors mpV (100 µM, lanes 5 and 6) or 3,4-dephostatin (100 µM, lanes 10 and 11) also show tyrosine phosphorylation of P2X₇ subunit in the presence of the phosphatase inhibitors (lanes 5 and 10), which is reduced when BzATP is added prior to application of phosphatase inhibitor (lane 11), but not when BzATP is added after the phosphatase inhibitor (lanes 3 and 6). (C) Tyrosine phosphorylation occurs on P2X₇ receptor but not P2X₂ receptor. Similar experiment to others performed on HEK cells transiently transfected with P2X₇-EE (lanes 1 and 2) or P2X₂-EE (lanes 3 and 4) receptors. Immunoprecipitation was with anti-EE Ab in the absence or presence of bpV as indicated. Anti-PY20 blotting detected phosphotyrosine-P2X₇ after bpV treatment (b, lane 2) but no tyrosine phosphorylation of P2X₂ receptor (b, lanes 3 and 4). Stripping and re-probing with C-terminal anti-P2X₇ (a, lanes 1 and 2) or anti-P2X₂ (a, lanes 3 and 4) confirms tyrosine phosphorylation of P2X₇ but not P2X₂ receptor even though there is greater expression of P2X₂ protein.