Fig. 3. ATP-induced CREB phosphorylation was transient compared to that induced by forskolin.

(A) Western blots showing time-course of CREB phosphorylation in the presence of forskolin (50 μM) and ATP (10 μM). Blots were probed with an antibody against CREB phosphorylated at serine 133 (top), stripped, and reprobed with an antibody against total CREB (bottom). (B) Analysis of four experiments. For each experiment, the magnitude of the Phospho-CREB:total CREB ratio in the presence of ATP or forskolin minus the unstimulated value was set to 100%. CREB phosphorylation at 1 hr and 2 hr is expressed as a percentage of this 15 min value. Although both forskolin and ATP rapidly raise phospho-CREB within 15 min (A), forskolin induced CREB phosphorylation remained significantly elevated up to 2 hr, whereas ATP-induced phospho-CREB levels returned to baseline levels by 1 hr. *, p < 0.0001 for forskolin vs. ATP at 1 hr (n = 5 experiments) and at 2 hrs (n = 4 experiments). (C) Western blot showing Ca²⁺-dependence of ATP-induced CREB phosphorylation in astrocytes. Cells were pretreated for 30 min with BAPTA-AM (150 μM) and then treated with vehicle (DMSO), forskolin (50 μM) or ATP (10 μM) for 15 min as indicated.