Fig. 8.

P2RX7 agonist-induced CREB/CBP complex formation. (A) RAW 264.7 macrophages were treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for the indicated time-points. Cells were lysed with 3× GST lysis buffer, and equal amounts of protein were incubated with GST-KIX beads for 18 h. The proteins that were pulled down with the GST-KIX beads were immunoblotted for CREB. A representative blot from three independent experiments is shown. (B) RAW 264.7 macrophages were pretreated for 15 min with vehicle (DMSO) or 10 μM UO126 and then were treated with vehicle (2.5 mM HEPES) or 250 μM BzATP for the indicated time-points at 37°C. Cells were lysed with 3× GST lysis buffer, and equal amounts of protein were incubated with GST-KIX beads for 18 h. Pulled-down proteins were immunoblotted for CREB. The results of independent experiments (n=3- to 5-min treatment; n=4 for 15-min treatment) were collated and are represented as a percent of BzATP-induced CREB phosphorylation (mean±sem); *, P < 0.0005.