Fig. 7: Effects of causative rs736799474 on enhancer activity and binding of heterophil nuclear proteins.

a Dual-luciferase reporter assay concerning the effect of the rs736799474 [T] to [C] transition on activity of a PTPRJ enhancer. Fragments containing the candidate variations were synthesized: Fragment 1 containing allele C, and Fragment 2 containing allele T. Each fragment was cloned into the 5’ end of a pGL4.18 vector for analysis of enhancer activity (enhancer-D). Empty pGL4.18 (Basic) was used as a negative control, and pGL4.18 containing the PTPRJ promoter (PTPRJ Promoter) was used as a reference for enhancer activity. Data represent the mean ± SD from three biological replicates per vector. Activities of the pGL4.18 + C and pGL4.18 + T vectors were compared using Student’s t-test. ** indicates P < 0.01, * indicates P < 0.05. b Effects of rs736799474 alleles on binding of heterophil nuclear proteins. EMSA was performed using biotin-labeled oligo probes containing either rs736799474 allele (C) or allele (T). Two microliters of nuclear proteins (5 μg/μl) from heterophils were incubated with 40 pmol of biotin-labeled probes. Specific binding was confirmed using 200-fold excess unlabeled cold probes containing the appropriate rs736799474 allele (C cold probe or T cold probe). Arrows indicate changes in protein binding capacity between alleles.