FIG. 8.

Effect of calcium and cGMP on CREB phosphorylation and effect of Cam-kinase and MAP kinase inhibitors on calcium- and cGMP-stimulated transcription. (A) UMR106 cells (left panel) and C6/GKII.1 cells (right panel) were treated for 1 h with 0.3 μM A23187 (lane 2), 250 μM CPT-cGMP (lane 3), or both (lane 4). Equal amounts of cell extracts were analyzed by SDS-PAGE and Western blotting with an antibody specific for Ser¹³³-phosphorylated CREB (pCREB, upper panel) and an antibody that recognizes CREB irrespective of its phosphorylation status (CREB, lower panel). The phospho-CREB antibody cross-reacts with phosphorylated ATF-1 and probably CREM (64). (B) UMR106 cells were transfected with pCRE-Luc, pRSV-βGal, and G-kinase II and treated with A23187 (Ca⁺⁺), CPT-cGMP (cGMP), or both, as described in the legend to Fig. 3. At 24 h before harvesting, some cells were treated with 0.1% dimethyl sulfoxide (vehicle; DMSO), 10 μM U0126, 10 μM SB20358, or 10 μM KN62. Reporter gene activities were normalized as described for Fig. 3.