conjugated secondary antibody, followed by exposure to autora- diography film. The protein band intensities were quantified by densitometry. Co-immunoprecipitation studies Untreated and IL-13-treated HASM cells were prepared for co- immunoprecipitation studies under native conditions in order to preserve protein-protein associations. After indicated treatment, cells were harvested and then lysed with lysis buffer. 1.5 mg of Protein G Dynabeads (Invitrogen) were incubated with 10 mg of anti-Gb rabbit IgG (Millipore) and incubated for 10 min at room temperature with rotation. Following several washes, the bound bead/antibody complex was added to sample, mixed by pipetting, and incubated for 2 hr at 4uC with rotation. The captured bead/ Protein G/antigen complex was then washed several times and eluted at pH 3.0 with rotation at room temperature. The precipitated immunocomplexes were subsequently analyzed by immunoblotting using anti-phospho-c-SrcTyr416 antibody. Expression of PDE4D mRNA transcripts After attaining confluence, the cells were starved in unsupple- mented Ham’s F12 media for 24 hr, subsequently treated with IL- 13, and then examined under different experimental conditions for induced changes in PDE4D mRNA expression, determined by RT-PCR as previously described [10,11,23]. Detection of PDE4D mRNA transcripts Total RNA was extracted from untreated and IL-13-treated HASM cells, in the absence and presence of co-treatment with specific inhibitors, using the TRIzol method (Invitrogen). cDNAs were then isolated by RT-PCR using the SuperScript First Strand Synthesis System kit from Invitrogen, with the following oligonucleotide primer sets (Integrated DNA Technologies): for PDE4D, 59-CGGAGATGACTTGATTGTGAC-39 (forward) and 59-CGTTCCTGAAAAATGGTGTGC-39 (reverse); and for b-actin, 59-GAGAAGAGCTACGAGCTGCCTGAC-39 (forward) and 59-CGGAGTACTTGCGCTCAGGAGGAG-39 (reverse). The reaction volume was 20 ml and cycling conditions used were 35 cycles of 30 sec denaturation at 95uC, followed by 30 sec annealing at 60uC and elongation at 72uC for 30 sec. Ex-Tag (Takara Biotechnology) was used as DNA polymerase. Statistical analyses Results are expressed as mean 6 SE values. Comparisons between groups were made using the Student t test (2-tailed), References 1. Druey KM (2009) Regulation of G-protein-coupled signaling pathways in allergic inflammation. Immunol Res 43: 62–76. 2. Hepler JR, Gilman AG (1992) G proteins. Trends Biochem Sci 17: 383–387. 3. Goldsmith ZB, Dhanasekaran DN (2007) G protein regulation of MAPK networks. Oncogene 26: 3122–3142. 4. Gerthoffer WT, Singer CA (2003) MAPK regulation of gene expression in airway smooth muscle. Respir Physiol Neurobiol 137: 237–250. 5. Hakonarson H, Grunstein MM (2003) Autocrine regulation of airway smooth muscle responsiveness. Respir Physiol Neurobiol 137: 263–276. 6. 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Statistical analyses were conducted by using the Prism computer program by Graph Pad Software Inc. Supporting Information Figure S1 Inhibition of Gbc signaling prevents induced changes in agonist responsiveness in OVA serum- sensitized rabbit ASM tissues. Relative to untreated (vehicle-exposed) controls, ASM tissues exposed to control serum exhibit similar Tmax responses to ACh (A) and Rmax responses to isoproterenol (B) both in the absence and presence of pre- treatment with either MPS peptide alone or anti-Gbc blocking peptide. By comparison, OVA serum-exposed ASM tissues exhibit significantly increased Tmax responses (A) and reduced Rmax responses (B) that are prevented by pre-treatment with anti-Gbc blocking peptide, whereas pre-treatment with MPS alone has no effect. Data are mean 6 SD values from 4–7 experiments. Treated tissues are compared to untreated (vehicle-exposed) controls using unpaired two-tailed Student t-test. *p,0.05; **p,0.01. (TIF) Figure S2 Anti-Gbc blocking peptide prevents in vivo antigen-induced airway hyperresponsiveness in OVA- sensitized rabbits. Relative to OVA-challenged control (non- sensitized; n = 4) rabbits, MCH-induced decreases in Cdyn are significantly enhanced at 24 hr following antigen challenge in OVA-sensitized rabbits (n = 4). This heightened bronchoconstric- tor responsiveness to MCh is suppressed in OVA-sensitized rabbits that are treated either with inhaled anti-Gbc peptide (1 mg/Kg; n- = 4) or budesonide (0.5 mg/Kg; n = 3) prior to antigen challenge. Note: Data represent Cdyn responses associated with corresponding Rrs responses shown in Fig. 2. Data are mean 6 SE values. ANOVA used for multiple comparisons of mean Rrs values. *p,0.05; **p,0.01. (TIFF) Author Contributions Conceived and designed the experiments: MMG GN. Performed the experiments: AH GN JSG MMG JM JKC PK MJ. Analyzed the data: MMG GN JM JKC PK. Contributed reagents/materials/analysis tools: MMG JSG. 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Koch WJ, Hawes BE, Allen LF, Lefkowitz RJ (1994) Direct evidence that Gi- coupled receptor stimulation of mitogen-activated protein kinase is mediated by Gbc activation of p21ras. Proc Natl Acad Sci USA 91: 12706–12710. 15. Luttrell LM, Hawes BE, van Biesen T, Luttrell DK, Lansing TJ, et al. (1996) Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbc subunit- mediated activation of mitogen-activated protein kinases. J Biol Chem 271: 19443–19450. PLoS ONE | www.plosone.org 14 February 2012 | Volume 7 | Issue 2 | e32078