Presentation of the A4GALT gene, 3 candidate regulators, and gel shift assay showing no binding of KLF1 to the P¹-specific rs5751348G sequence. (A) Schematic representation of A4GALT with the 3 SNPs suggested to determine the P₁/P₂ phenotypes. The main candidate SNP is marked with a black circle, with the others in white. Gray boxes depict noncoding exons with the open reading frame (ORF) in red and introns represented by horizontal black lines. In the lower, magnified section of exon 2a and the following intron, the P¹-associated variants of each SNP and putative binding sites for selected TFs are indicated in boldface type. The main SNP (rs5751348) is highlighted in blue (P¹ allele) and red (P² allele). (B) EGR1, RUNX1, and KLF1 sequence logos showing binding preferences are adapted from the JASPAR database.²⁵  The sequence logos for EGR1 and KLF1 are reversed to match the reading direction of A4GALT. Murine binding data are used for RUNX1 and KLF1 because data sets for the human TF are scarce (RUNX1) or not available (KLF1) in the database. For more data on RUNX1-binding requirements, see supplemental Figure 2. Bottom, 3 graphs highlighting the relative score of the binding energy for the 3 candidate TFs are displayed for both P¹ and P² alleles (G and T). The scale of the x-axis is centered around the default binding threshold of 0.8. (C) Oligonucleotides representing the sense strand in the probes used are shown with the nucleotide corresponding to rs5751348 with the P¹ variant in blue and P² in red. (D) EMSA shift and supershift reaction using P¹/P²-32 probes, with nuclear extracts (NEs) from HEL cells or recombinant KLF1 and anti-KLF1. (E) Blotting with anti-KLF1 on protein pull-downs with P¹ probes of various lengths, P²-37 probe and a scrambled version of the P¹-37 probe. NE are shown as positive control for antibody binding and blotting of a serial dilution of recombinant KLF1 for anti-KLF1 to show appropriate detection by the antibody under the conditions used. Differences in molecular weights of KLF1 are due to an attached tag on the recombinant version of the protein.