CREB Activates BMP2 Expression in Osteoblasts Figure 5. CREB transactivates BMP2 through CRE in the BMP2 promoter. (A) BMP2 promoter reporter activity in C2C12 cells, co-transfected with CREB plasmid and a series of truncated reporter constructs for 24 hours, was measured with b-gal normalization. (B) Sequence of 2150/+165 of mouse BMP2 promoter. Putative sequences for CRE1, CRE2 and CRE3 are underlined and bolded. Sequences of ChIP primers are underlined only. (C) Sequences of mutant CRE1, CRE2 and CRE3 (mCRE1, mCRE2 and mCRE3). (D) BMP2 promoter reporter activity in C2C12 cells, co-transfected with wild-type 2150/+165-Luc (WT) and mutant 2150/+165-Luc in which CREs were mutated (mCRE1, mCRE2 and mCRE2), and with CREB plasmid for 24 hours, was measured. * p,0.01 (CREB transfection on mCRE2 vs WT or mCRE1 or mCRE3 reporters; mean6SE, n = 8). (E) Effects of PTH on BMP2 promoter activity. C2C12 cells were co-transfected with wild-type 2150/+165-Luc (WT) and mutant 2150/+165-Luc in which CRE2 was mutated (mCRE2) and treated with PTH at 50 nM for 24 hours. * p,0.01 (PTH mCRE2 vs PTH on WT reporter; mean6SE, n = 8).F) ChIP assay. Nuclear DNA- protein complexes were extracted from C2C12 cells treated with PTH at 50 nM for 6 hours and precipitated with anti-CREB antibody. PCR was PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e20780