Quantifying BMP Signaling in C2C12BRA Cells

C2C12BRA cells were stably transfected with a BMP-responsive, Id1 promoter, firefly luciferase reporter gene construct to assess BMP signaling, as previously described^{54 (link),55 (link)}. Lipofectamine (Invitrogen) was used to transfect cells containing the reporter gene with wild-type ALK6, rs34970181 (R371Q) ALK6, or constitutively active (Q204D) ALK6 (kindly provided by Dr. Takeshi Imamura, JFCR Cancer Institute, Tokyo, Japan). Transfection efficiency was determined using pRL-TK renilla luciferase (Promega). The ratio of firefly to renilla luciferase activities was calculated to quantify the amount of BMP signaling normalized to transfection efficiency. For BMP stimulation experiments, transfected C2C12BRA cells were incubated for 16 hours in serum-free media together with 0, 5, or 25 ng/ml of BMP2.