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 4 Enlarged

Fig. 4:

EMSA analysis confirms results obtained through WEMSA.EMSA using labeled Sp-1-like oligo spanning positions -91 to -79 of the CD14 promoter. Serum starved THP-1 cells were either untreated or treated with 100 nM calcitriol for 30 min followed by preparation of nuclear extracts for EMSA as described in Materials and Methods. Lane 1, free labeled oligo. Lane 2, nuclear extract of untreated cells combined with Sp-1-like oligo. Lane 3, nuclear extract of calcitriol-treated cells combined with Sp-1-like oligo. Lane 4 represents nuclear extract from untreated cells combined with Sp-1-like oligo and unlabelled excess of Sp-1-like oligo.

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