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Suppl. 1. Levels of endogenous Bsp by immunostaining. Osx-null calvarial cells transfected with Flag-HA-Osx for 24h were fixed with 4% formaldehyde for 20min at room temperature, permeabilized with 0.1% triton X-100 for 10min, blocked with 3%BSA and then incubated with primary antibodies anti-HA (A) and anti-Bsp (B) for 1 hour in humidified chamber followed by detection with alexa-fluor conjugated secondary antibodies. Cells were visualized under microscope with 40X objective. Panel C indicates DAPI staining to visualize nuclei, and Panel D indicates a merged of three colors.

Suppl. 2 and 3. DNA methylation in the Bsp promoter persists in Osx-null calvaria. Sequencing chromatogram of PCR products of region II and III amplified from bisulfite-treated DNA isolated from ES cells, Osx-wt and Osx-null calvaria of embryos E18.5. Arrow indicates methylated cytosines (8-14) undergo DNA demethylation in wild type when compared with that in ES cells and Osx-null calvaria.

Primers sequence used in this study

jbmr2103-sm-0002-SuppFigs.pdf206KSupplemental Figures

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