Identification of a Calcitriol-Regulated Sp-1 Site in the Promoter of Human CD14 using a Combined Western Blotting Electrophoresis Mobility Shift Assay (WEMSA)

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Author: Moeenrezakhanlou, A
Author: Reiner, NE
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Methods Article

Identification of a Calcitriol-Regulated Sp-1 Site in the Promoter of Human CD14 using a Combined Western Blotting Electrophoresis Mobility Shift Assay (WEMSA)

Alireza Moeenrezakhanlou¹, Devki Nandan² and Neil E. Reiner²^*

¹ Department of Microbiology and Immunology, University of British Columbia, Faculties of Medicine and Science, and Vancouver Coastal Health Research Institute (VCHRI), Vancouver, British Columbia, Canada, V5Z 3J5 and School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.
² Department of Microbiology and Immunology, University of British Columbia, Faculties of Medicine and Science, and Vancouver coastal health Research Institute (VCHRI), Vancouver, British Columbian, Canada, V5Z 3J5.

* To whom correspondence should be addressed: Neil E. Reiner, Department of Microbiology and Immunology, University of British Columbia, Faculties of Medicine and Science, and Vancouver Coastal Health Research Institute (VCHRI). Division of Infectious Diseases, University of British Columbia, Rm 452D, 2733 Heather Street, Vancouver, BC. Canada. Phone: 604-875-4011. Fax: 604-875-4013. Email: ethan@interchange.ubc.ca

Biol. Proced. Online 2008;10:29-35. doi:10.1251/bpo140
Submitted: March 23, 2007; Accepted: December 12, 2007; Published: February 17, 2008.

Indexing terms: Electrophoretic Mobility Shift Assay; Transcription Factors; Antigens, CD14.

Abbreviations: TF, transcription factor; VDRE, vitamin D response element; WEMSA, western blot electrophoretic mobility shift assay.

Figure 2 Enlarged

Fig. 2:

WEMSA shows that Sp-1 binding to a specific site in the CD14 promoter is regulated by calcitriol. Serum starved THP-1 cells were either treated or not with 100 nM calcitriol for 30 min followed by preparation of nuclear extracts for WEMSA as described in Materials and Methods. Nuclear extracts were incubated with four unlabelled oligos denoting putative Sp-1-like transcription factor-binding sites in CD14 promoter (A) and DNA protein complexes were separated by non-denaturing gel electrophoresis followed by transfer to nitrocellulose for Western blotting for Sp-1 (B). The results shown are from one of three independent experiments that yielded similar results.

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