[Abstract]  [Full Text]  [PDF]

Alireza Moeenrezakhanlou¹, Devki Nandan² and Neil E. Reiner^2*

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.

Calcitriol (1α, 25-dihydroxyvitamin D₃) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot analysis (WEMSA). Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position -91 to -79 (relative to the transcription start site), bound the transcription factor Sp1. Sp-1 binding to this site was demonstrable using nuclear extracts from control cells. Notably, binding activity was attenuated in nuclear extracts prepared from cells that had been incubated with calcitriol, thus suggesting Sp-1 involvement in calcitriol induction of CD14 expression. Notably, these results show that like EMSA, WEMSA can be broadly applied to aid in the identification of transcription factors involved in regulating gene expression. WEMSA, however, offers a number of distinct advantages when compared with conventional EMSA. Antibodies used for WEMSA often provide less ambiguous signals than those used in EMSA, and these do not have to recognize epitopes under native conditions. In addition, WEMSA does not require the use of labeled oligos, thus eliminating a significant expense associated with EMSA.