Binding of transcription factor SP1 to G allele of SNP rs2596538.

(A) Multiple alignment of a GC box and DNA sequence of A or G probe of SNP rs2596538 used in EMSA. (B) EMSA using the labeled G allele of SNP rs2596538 and nuclear extract from heat treated HLE cells. Non-labeled consensus oligonucleotides of seven transcription factors are used as competitors. Pointed arrow indicates shifted band. (C) EMSA using the labeled G allele of SNP rs2596538 and nuclear extract from heat shock treated HLE cells in the presence of anti-SP1 antibody or normal rabbit IgG. Asterisks on the left side indicate the shifted (*) and super-shifted bands (**). Normal rabbit IgG serves as a negative control. (D) ChIP assay using HepG2 and HLE cell lines were ectopically expressed with SP1 protein. DNA-protein complex was immunoprecipitated with anti-SP1 antibody followed by PCR amplification using a primer pair flanking SNP rs2596538. DNAs precipitated without antibody are served as a negative control. PCR primers flanking the 3' UTR region of MICA are served as a negative control. (E) Genotype distribution at SNP rs2596538 in PCR fragment amplified from the input genomic DNA and DNA-protein complex immunopurified from HepG2 cells by using anit-SP1 antibody. *P<0.05 by Student's t-test.