Fig. 7.

Effect of calcium chelator BAPTA on P2RX7 agonist-induced ERK/CREB phosphorylation. (A) RAW 264.7 macrophages were loaded with vehicle (DMSO) or BAPTA-AM at increasing concentrations (1 μM–30 μM) for 30 min at 37°C. Following inhibitor incubation, the cells were washed twice and treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for 5 min and immunoblotted for ERK1/2 phosphorylation. (Note: BzATP-induced ERK1/2 phosphorylation without BAPTA-AM was done in duplicate.) Immunoblotting was performed with anti-Grb2 antibody as a loading control. The results of three independent experiments were combined and are represented as a percent of BzATP-induced ERK1 (left panel) and ERK2 (right panel) phosphorylation (mean±sem). (B) RAW 264.7 macrophages were loaded with vehicle (DMSO) or BAPTA-AM at increasing concentrations (1 μM–30 μM) for 30 min at 37°C. Following inhibitor incubation, the cells were washed twice and treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for 5 min and immunoblotted for pCREB/pATF1. Immunoblotting was performed with anti-Grb2 antibody as a loading control. The results of three independent experiments were collated and are represented as a percent of BzATP-induced CREB phosphorylation (mean±sem); *, P < 0.01; **, P < 0.05; ***, P < 0.003.