Effect of RUNX1 overexpression and RUNX1 siRNA downregulation on RAB1B protein expression and promoter activity. (A) Immunoblot analysis of RUNX1, RAB1B, and actin on overexpression of RUNX1 in HEL cells. Bars shown are an average of 3 independent experiments. P values shown are for comparison with control (pCMV6 vector alone). (B) Effect of RUNX1 overexpression on RAB1B promoter activity. WT construct and mutant constructs with sites 1-4 individually mutated were cotransfected with RUNX1-pCMV6 expression vector (green bars), empty vector pCMV6 (blue bars), or neither (red bars) in HEL cells. Reporter activity was measured at 48 hours. Bar graphs show activity as mean ± SE of 3 independent experiments in triplicate. (C) Immunoblot analysis of lysates from HEL cells transfected with RUNX1 siRNA showing inhibition of RAB1B protein. Bars shown are average of 3 independent experiments. P values are for comparison with control vector. (D) Effect of RUNX1 siRNA on RAB1B promoter activity. RAB1B WT promoter construct was cotransfected with control siRNA or RUNX1 siRNA in HEL cells. Reporter activity was measured at 48 hours. Bar graphs show activity as mean ± SEM of 3 independent experiments in triplicate. RUNX1 siRNA reduced RAB1B protein and RAB1B promoter activity.