Preparation of an anti-Cdx-2 antibody for analysis of different species Cdx-2 binding to acat2 promoter.
The homeodomain protein, Cdx-2, as transcription factor has been implicated in the transcriptional regulation of genes expressed in small intestine and the process of tumorgenesis. In current work, a conserved mouse Cdx-2 domain (mCdx-2D) coded by its cDNA fragment, which was amplified and cloned into the expression vector pGEX-4T1, was expressed as a fusion protein with GST (GST-mCd x-2D) and purified by one step of affinity chromatography. A polyclonal antibody against Cdx-2 was raised by using the recombinant fusion protein GST-mCdx-2D as antigen and was fractionated from the rabbit anti-serum. Western blot and EMSA (electrophoretic mobility shift assay) demonstrate that the natural and denatured Cdx-2s from different species (mouse and human) can be detected by the prepared anti-Cdx-2 antibody. Most notably, we found that the Cdx-2 in human intestine cell line Caco-2 is expressed in a differentiation-dependent manner and can efficiently bind to the mouse and human acat2 (acyl-coenzyme A: cholesterol acyltransferase 2) promoter regions, suggesting that the transcriptional factor Cdx-2 may play a role in regulating the acat2 expression in the intestinal cells.
Song
Bao-Liang
BL
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China.
Qi
Wei
W
Wang
Can-Hua
CH
Yang
Jin-Bo
JB
Yang
Xin-Ying
XY
Lin
Zhi-Xin
ZX
Li
Bo-Liang
BL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity; D001665:Binding Sites / Q000235:genetics; D000071616:CDX2 Transcription Factor; D016466:CHO Cells; D018938:Caco-2 Cells; D006224:Cricetinae; D015342:DNA Probes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D005982:Glutathione Transferase / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D002785:Sterol O-Acyltransferase / Q000235:genetics*; D015534:Trans-Activators; D014407:Tumor Cells, Cultured; D000097587:Sterol O-Acyltransferase 2
D000911:Antibodies, Monoclonal; D000071616:CDX2 Transcription Factor; D015342:DNA Probes; D005982:Glutathione Transferase; D018398:Homeodomain Proteins; D011993:Recombinant Fusion Proteins; D002785:Sterol O-Acyltransferase; D015534:Trans-Activators; D000097587:Sterol O-Acyltransferase 2