Effect of RUNX1 overexpression and RUNX1 depletion by siRNA on RAB31 protein expression and promoter activity. (A) Effect of RUNX1 overexpression. Immunoblotting of RUNX1 and RAB31 of HEL lysates transfected with RUNX1 expression plasmid (RUNX1-pCMV6) and empty plasmid (PCMV6). Actin was the loading control. Presented as mean ± standard error of the mean (SEM) of 3 independent experiments. (B) Effect of RUNX1 overexpression on promoter activity (luciferase). RAB31 WT promoter construct was cotransfected with RUNX1 expression plasmid or empty plasmid in HEL cells. Promoter activity was enhanced with RUNX1 plasmid. Activity shown as mean ± SEM of 3 independent experiments in triplicate. (C) Effect of RUNX1 downregulation (siRNA). Immunoblotting of HEL lysates transfected with control or RUNX1 siRNA. Actin was the loading control. Presented as mean ± SEM of 3 independent experiments. (D) Effect of RUNX1 siRNA on promoter activity. RAB31 WT promoter construct was cotransfected with control or RUNX1 siRNA. Promoter activity was reduced with RUNX1 siRNA. Presented as mean ± SEM of 3 independent experiments in triplicate. RUNX1 siRNA inhibited RAB31 protein expression and promoter activity. NS, not significant.