formaldehyde for 10 minutes to cross-link the chromatin. The ChIP assay was performed following the protocol of the ChIP kit (Upstate, MA). Briefly, cells were scraped and sonicated on ice to shear chromatin DNA down to 0.2–1.0 kb fragments. The sonicated cell supernatants were pre-cleared with a Protein A agarose/salmon sperm DNA slurry. Anti-CREB antibody (Cell Signaling Technology, Inc.) was added to the supernatant at 4uC overnight, followed by incubation with fresh Protein A agarose beads for 1 hour at 4uC for precipitation. The specific protein- DNA complex was disassociated and DNA fragments were purified. With these DNA fragments as templates, PCR was performed using primers F-59ggcatcgcccacgatggctg and R-59caa- gttcaagaagtctcc, to amplify a sequence containing a specific CRE in the BMP2 promoter. The BMP2 promoter DNA input was used as a positive control. Goat IgG served as a negative control. Electrophoresis mobility shift assay (EMSA) C2C12, 2T3, and MC3T3-E1 cells (approximate 2–46106) were collected by digestion, and the nuclear extracts were prepared using a kit (Pierce). A synthesized, biotin-labeled and annealed DNA probe (59tggtccctgaggccgacgacagcagcagccttgc) at 20 fmols, which contained the CRE2 binding site sequence in the BMP2 promoter, was reacted with nuclear extracts for 20 minutes. The reactions were applied onto 5% polyacrylamide gels (Bio- Rad), and then transferred to Nylon membranes. Biotin-labeled DNA was detected using a streptavidin-horseradish peroxidase conjugate and chemiluminescent substrate (Pierce). To examine super shift of DNA-protein complexes, an antibody against CREB was added to DNA-protein binding reactions and loaded to the gels. The detailed methods followed the protocol of LightShift Chemiluminescent EMSA Kit (Pierce). References 1. Iida-Klein A, Zhou H, Lu SS, Levine LR, Ducayen-Knowles M, et al. 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C2C12 cells were treated with IBMX at 0 to 300 mM for 1 hour (Cq); IBMX at 50 mM for 0 to 4 hours (Dq); or KT5720 at 0 to 10 mM for 1 hour (Eq); KT5720 at 10 mM for 0 to 2 hours (Fq). Phospho-CREB was detected by Western blot. The signal intensity of Western blot band was quantitated using GelScan v 5.1 (BioSciTech) with normalization by the intensity of b-actin. (TIF) Acknowledgments We thank Dr. Richard Goodman for providing CREB expression plasmid and CREB responsive reporter construct, Dr. Stephen Harris for providing BMP4 promoter reporter construct, and Dr. Xiangli Yang for providing ATF4 expression plasmid. We thank Dr. Theo Mantamadiotis for providing CREBfloxed mice and Dr. Gerard Karsenty for permission to use Col1a12.3-Cre mice to generate the conditional CREB knockout mice. The corresponding author Dr. Ming Zhao sincerely thanks the late Dr. Gregory Mundy for his scientific advice for this study. Author Contributions Conceived and designed the experiments: MZ. 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