Bcr and Abr Regulate PH Figure 6. Enhanced phosphorylation of p38 and IL-6 produc- tion in vivo and in mutant PASMC. A, Representative image of Western blots analyzing the phosphorylation of p38 in the lungs from bcr2/2, abr2/2 and wt mice. B, Quantification of results for lungs from 6 mice/group. C–D, IL-6 was measured using an ELISA in serum (C) and lungs (D) of bcr2/2, abr2/2 and wt mice using 6 independent samples per genotype per condition. E–F, Western blot analysis of PASMC for p-p38 (E) and quantification (F) of results of 3 independent samples of PASMCs/group. G, IL-6 in PASMC supernatants after normoxia or hypoxia exposure. *p,0.05 compared with the values from the same genotype, or PASMCs in normoxia. #p,0.05 when compared with WT exposed to the same condition. doi:10.1371/journal.pone.0049756.g006 injection, animals were sacrificed and perfused with PBS through the right ventricle. The vena cava was cut to drain blood and PBS, thus allowing PBS to pass through the pulmonary and systemic circulation to flush out blood. After perfusion, lungs were harvested, blotted on gauze and weighted. Lungs were then placed in 1 ml of formamide and incubated at 56uC for 24 h for the extraction of Evans blue. The concentration of Evans blue was measured at OD620 and calculated against a standard curve. Pulmonary Arterial Smooth Muscle Cells Isolation and Culture PASMCs were isolated from the intrapulmonary arteries of male bcr2/2, abr2/2 and wt FVB/J mice as previously described [60]. In brief, mice were euthanized by CO2. The thorax and abdomen were rinsed with 70% ethanol. The intrapulmonary arteries were dissected with sterile scissors and forceps under microscope. The adventitia and intima were removed from the arteries. The arteries was cut into 1–2 mm long pieces and incubated with 1.5 mg/ml type II collagenase (Sigma) for 4–6 hours at 37uC with gentle shaking. After two washes with culture medium (DMEM with 10% fetal bovine serum, 1% penicillin/ streptomycin, 1% glutamine; Invitrogen), PASMCs were collected Figure 7. Schematic representation of the signal transduction pathways affected in PASMC lacking Abr and Bcr. Hypoxia directly or indirectly activates a G-nucleotide exchange factor (GEF) for Rac, resulting in elevated levels of GTP-bound Rac. In the absence of Bcr or Abr, Rac is not downregulated to its inactive GDP-bound form and this causes prolonged activation of p38 MAPK and production of IL-6. The inhibitor Z62954982 prevents the generation of the GTP-bound Rac that switches these pathways on, by interfering with the binding of Rac to the GEF that is chronically switched on in hypoxia. doi:10.1371/journal.pone.0049756.g007 PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49756