Involvement of CREB (Ser133) in the induction of FcγRII transcription. (A) Representative Western blot analysis of phospho-CREB (Ser133) or total CREB in nuclear extracts from PLB cells, PLB-D cells, or PLB-D cells with daily addition of 12 μM PGE₂ during 4 days of differentiation by 1,25(OH)₂D₃. The intensity of each band of phospho-CREB was divided by the intensity of each band of CREB after quantification by densitometry scanning. (B) Representative EMSA for DNA-binding activity of CREB. Nuclear extracts isolated from PLB cells during 4 days of differentiation by 1,25(OH)₂D₃ were incubated with ³²P-labeled probe containing CREB consensus sequence from FcγRIIA promoter. For competitive inhibition assay, 50-fold molar excess of unlabeled CREB antibodies against CREB or labeled CREB mutant probe was added. (C) EMSA for DNA-binding activity of CREB was not detected during differentiation by 1,25(OH)₂D₃ in PLB-D cells and in parent PLB cells in the presence of 30 μM indomethacin. DNA-protein complexes were analyzed on a 7% nondenaturing polyacrylamide gel. Three other experiments showed similar results.