Fig. 5

Functional analysis of − 13838G > A as enhancer in Caco-2 cells. a The luciferase reporter assays of LCT promoter and enhancer constructs. As a control, cells were transfected with the promoterless pGL3-basic vector. Basal levels of expression were assessed using hLPH1085 constructed with a pGL3-basic vector and 1085 bp LCT promoter. Three different enhancer haplotypes were inserted upstream of the hLPH1085. Differences in expression of the reporter gene were indicated by the relative luciferase activities. b ChIP-qPCR for the DNA–protein complex enriched by anti-HNF4A antibody. The complex enriched by anti-IgG antibody was amplified as the control. c Electrophoretic mobility shift assays with Caco-2 cell nuclear extracts and oligonucleotide probes spanning − 13838G > A SNP. Specific competitors: − 13838*A (lane 4) or − 13838*G (lane 9) probes without biotinylation; unspecific competitor: unlabeled oligonucleotide probe in which the 8 bp nucleotides surrounding − 13838G > A were mutated (lanes 5 and 10). d The luciferase reporter assays for caco-2 cells co-transfected with the LCT promoter and enhancer constructs harboring − 13838*A or − 13838*G in the presence of HNF4A (hLPH1085 − 13910C − 13838A/G + HNF4A) and pcDNA3.1 expression constructs (hLPH1085 − 13910C − 13838A/G + pcDNA3.1). Statistical significance was tested using the unpaired Student’s t-test: *0.01 < P < 0.05; **P < 0.01