Figure 1.

Characterisation of rs2069837 and detection of binding proteins. (A) rs2069837 (hg19, chr7: 22768026) is located in the second intron of IL6. Epigenetic markers (DNase and H3K27ac) in multiple immune cells indicate that this locus is close to a functional region. (B) EMSA assay using nuclear protein from HEK293 cells demonstrating that more DNA–protein complexes were formed with the probe with allele A (lane 4) than allele G (lane 8); both unlabeled probes with A allele or G allele (24 pmol, 200×) can totally compete out the corresponding shifted band separately (lane 5 and lane 9); unlabeled G allele probe cannot completely compete out the shifted band formed with the A allele probe (lane 6); unlabeled A allele probe was able to compete out the shifted band formed in the presence of the G allele probe (lane 10). (C) Different concentrations of the unlabeled A allele probe were applied in the competitive EMSA assay (1×, 10×, 50×, 100×, 200×, 400×). At 10× concentration, the band can be competed out (lane 6). (D, E) Validation using Western blotting. More HDAC and MFE2 proteins bound to the probe with A than with G allele, while no differences were detected in TAF1, ARID3A, GATA1 and GATA2 between probes with A and G alleles. When the four nucleotides around rs2069837 were mutated, the binding of those proteins decreased except for ARID3A. The protein binding in the unlabeled group was barely observed. A, probe with A allele; EMSA, electrophoretic mobility shift assay; G, probe with G allele; Mu, probe with mutated sequence; NL, non-biotinylated oligos with A allele; LC, loading control.