Biochemical and Biophysical Research Communications
Calcineurin regulates phosphorylation status of transcription factor osterix
Section snippets
2. Materials and methods
Plasmid construction and stable cell lines. Mouse osterix full length cDNA was cloned from total cellular RNA of mouse osteoblastic MC3T3-E1 cells by RT-PCR as previously reported [12]. Osterix cDNA was subcloned into pcDNA3.1-Flag (modified pcDNA3.1, Invitrogen, Carlsbad, CA, USA) and pEGFP-C3 (BD Biosciences, Palo Alto, CA, USA) expression vectors, and confirmed by sequencing. To generate Flag-OsxΔVP (carrying a deletion of valine and proline at position 109 and 110, respectively), we carried
Calcineurin associates with osterix
To investigate whether calcineurin could interact with osterix, proteins prepared from HEK-Flag-Osx and HEK-Flag cells were immunoprecipitated with Flag affinity gel, and subjected to Western blot analysis. Anti-calcineurin antibody interacted with Flag-osterix complex eluted from Flag affinity gel (Fig. 1A, upper panel, Flag-Osx). The molecular weight of a positive band was estimated as 61 kDa, corresponding to that of the calcineurin. Anti-calcineurin antibody also recognized the protein in
4. Discussion
To identify a new factor that could participate in osterix regulation, we used the cell lysates from HEK293 cells transfected with Flag or Flag-osterix plasmid and performed immunoprecipitation assay. We demonstrated that calcineurin associates with osterix in a direct manner, as determined by immunoprecipitation and Western analysis. The binding of calcineurin and osterix was further supported by their localization in nucleoplasm of HEK293 cells, determined by immunocytochemical study. Osterix
Acknowledgments
We thank Mrs. E. Sasaki for her skilful technical assistance. This study was supported in part by grants from the Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.
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