Figure 3.

Bone morphogenic protein (BMP) signaling and GRE–BMP-4 interaction. Reduced pSmad1/5/8 level in AdGRE animals at Day 7 (B and C) as compared with AdDL70 animals (A and C) by IHC and Western blot of lung protein lysate. The total Smad1 double band is indicated by an arrow in (C). pSmad1⁺ epithelial cells are outlined by arrows in (A and B). Please note the reduced overall staining intensity in the AdGRE animal (B) as compared with AdDL70 (A). (D) Semiquantitative densitometry of pSmad1/5/8 protein normalized to α-tubulin, expressed as n fold of pSmad1/5/8 protein versus AdDL70. (E) Reduced Id1 mRNA expression in lung homogenate of AdGRE animals, indicating reduced BMP-4 signaling, expressed as n fold mRNA versus AdDL70. (F) Reduced pSmad1/5/8 protein level in total protein lysate of A549 72 hours after AdGRE administration as compared with AdGFP (control). (G) Co-immunoprecipitation (IP) HA-tagged Gremlin and BMP-4 showing that an approximately 50-kD BMP-4 precursor protein interacts with Gremlin in AdGRE rat lungs at Day 7. α-Tubulin was used as loading control in (C and F). (A and B) Original magnification, 400×; scale bar, 20 μm. (D and E) Each bar represents the mean (±SEM) of three to six animals per group. *P < 0.05, **P < 0.01 versus AdDL70. IB, immunoblot.