Identification of RUNX1 as the P^1/P²-discriminating transcription factor binding to rs5751348G and its functional effect on A4GALT expression. (A) RUNX1 protein sequence, with all unique peptides identified by mass spectrometry marked in bold and underlined. (B) Blotting with anti-RUNX1 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. (C) EMSA shift competition assay with P¹-32 probe and increasing amounts of anti-RUNX1 to obtain blocking of shift. Relative quantification of KLF1 and A4GALT transcripts in siRNA-transfected MEG-01 (D) and HEL (E) cells. Relative quantification of RUNX1 and A4GALT transcripts in siRNA transfected MEG-01 (F) and HEL (G) cells. (D-G) Experiments were run in triplicate on 3 separate occasions. (H) Western blot of RUNX1 protein 48 hours after knockdown. The image displays 2 sections from the same blot, separated with a dashed line. (I) Quantification of the results in panel H, showing RUNX1 band intensity normalized to total protein content in lane. Error bars represent the standard error of the mean. *P < .05; **P < .01; ***P < .001 (Mann-Whitney U test). ns, not significant; RQ, relative quantity; siNeg Ctrl, negative control siRNA #1; siKLF1, 2 siRNAs targeting KLF1; siRUNX1, 2 or 3 siRNAs targeting RUNX1.