BMP9 activation of the BRE promoter is specific for ALK1. (A) NIH-3T3 cells were transiently transfected with pGL3(BRE)-luc and pRL-TK-luc and either pCDNA3 empty vector, pALK1, pALK2, pALK3, or pALK6 plasmids. After 4 hours, cells were treated with BMP9 (0.5 ng/mL) or BMP2 (50 ng/mL) for 15 hours. (B) Overexpression of the different human ALK receptor transcripts in transfected cells were controlled using semiquantitative RT-PCR. mHPRT was amplified to normalize for the amount of RNA used as starting material. (C) NIH-3T3 cells were transiently transfected with pGL3(BRE)2-luc and pRL-TK-luc. After 4 hours, cells were treated with BMP9 (0.5 ng/mL) or BMP2 (50 ng/mL) in the presence or absence of ALK1ecd or ALK3ecd (6-fold molar excess over respective BMP9 or BMP2 concentrations) for 15 hours. The luciferase activities were then measured as described in “Materials and methods.” The firefly luciferase activity was normalized to renilla luciferase activity. (A-C) Data show 1 representative experiment ± SD of 3.
