Characterization of RAB31 promoter. (A) RAB31 promoter region (−2023 bp from ATG codon) showing 4 RUNX1 consensus-binding sites. (B) RUNX1 binding to RAB31 upstream region by ChIP. PCR amplification of HEL cell chromatin immunoprecipitated by IgG (lane 1) and RUNX1 antibody (lane 2); PCR amplification of input or total DNA (lane 3) and amplification of genomic DNA (gDNA) (lane 4) using RAB31 primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control. (C) EMSA using WT nucleotide probes carrying RUNX1-binding site II (−957/−976; left) and site IV (−1997/−2016; right) in RAB31 promoter and PMA-treated HEL nuclear extracts. EMSA using site II probe (left panel) lane 1, no extract; lane 2, protein binding to the probe; lane 3, loss of protein binding by competition with excess unlabeled probe; lane 4, no loss of binding by competition with normal IgG; and lane 5, loss of protein binding on competition with anti-RUNX1 antibody. EMSA using site IV probe (right panel, lanes 1-5): similar results were obtained with the probe with site II. Representative of 3 independent experiments. (D) Luciferase reporter studies on RAB31 promoter in PMA-treated HEL cells. Left panel: WT RAB31 promoter with 4 RUNX1 sites (red boxes) and constructs with specific mutants (blue boxes). Right panel: shown is luciferase activity. Site II mutation shows increase in activity compared with WT construct; site IV mutation decreased activity, indicating sites II and IV are functional. Presented as mean ± standard error of the mean of 3 independent experiments in triplicate. NS, not significant.