Fig. 3

Antibody 3F6 has no effect on BMP-responsiveness in BMPR2 knock-down HEK293T cells. a Expression levels of endogenous BMPR2 in scramble control HEK293T cells (Control) and HEK293T cells carrying anti-BMPR2 shRNA (BMPR2-KD) compared to HPRT1 loading control. Data are expressed as normalized to scramble control (Control) using the 2^−∆∆Ct method. n = 6 per condition. Asterisk indicates p < 0.05 by unpaired t test. b, c Expression of endogenous BMPR2 in scramble control HEK293T cells (Control) and BMPR2-KD HEK293T cells compared to β-actin loading control. Approximate molecular weights are indicated in b. Representative immunoblot is shown in b and results from three independent runs are quantified in c (data are expressed as mean ± SEM ratio of BMPR2: β-actin normalized to scramble control (Relative Expression)). Asterisk indicates p < 0.05 by paired t test. d, e BMP2 induces phosphorylation of SMAD1, 5, and 8 (pSMAD1,5,8) in BMPR2-KD HEK293T cells. No effect on BMP2-responsiveness of BMPR2-KD HEK293T cells was observed with pre-treatment using control ascites (Ctrl Asc.) or 3F6. Approximate molecular weights are indicated in d. Results are quantified in E and expressed as mean ± SEM ratio of phosphorylated SMAD1,5,8: total SMAD1 relative to BMP2 treatment alone (Relative pS1:S1). n = 4 per condition