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. 2012 Sep 18;23(2):242–253. doi: 10.1038/cr.2012.135

Figure 2.

Figure 2

The CBS −4673 G allele attenuates transcription factor binding affinity. (A) SPR analysis comparing the binding affinity of nuclear extracts to DNA probes containing either the −4673 C or G alleles. The SPR-binding activity of the A-allele (|logKD| = 9.75 ± 0.65) was more than 1 000-fold higher than that of the C-allele (|logKD| = 6.12 ± 0.05). Each value represents the mean ± SD of three experiments. (B) Competition SPR assays performed in the presence of 5-fold excess non-biotinylated C- or G-allele probes. (C) ChIP assays using HEK293 and cardiovascular tissue samples. The presence of the SP1-binding CBS promoter was verified by PCR. (D) A luciferase construct containing either the C or G allele was co-transfected with pcDNA3.1 (control) or pcDNA3.1-SP1 expression plasmids. The actual values in HEK293 cells were as follows: pcDNA3.1-basic group: pGL3-basic = 0.34 ± 0.08, pGL3-C allele = 2.27 ± 0.16, and pGL3-G allele = 3.54 ± 0.23; pcDNA3.1-SP1 group: pGL3-basic = 0.43 ± 0.13, pGL3-C allele = 1.46 ± 0.12, and pGL3-G allele = 3.10 ± 0.16. The actual values in H9C2 cells were the following: pcDNA3.1 group: pGL3-basic = 0.31 ± 0.08, pGL3-C allele = 1.73 ± 0.14, and pGL3-G allele = 2.74 ± 0.10; pcDNA3.1-SP1 group: pGL3-basic = 0.27 ± 0.03, pGL3-C allele = 1.14 ± 0.12, and pGL3-G allele = 2.21 ± 0.18. Each value represented mean ± SD of three experiments, and each experiment was performed in triplicate.