G Protein bc-Subunit Signaling in Asthma Figure 3. Anti-Gbc blocking peptide suppresses pulmonary inflammation in OVA-sensitized rabbits. Relative to controls (A), lungs isolated from antigen-challenged OVA-sensitized rabbits exhibit diffusely scattered patchy foci of inflammatory cell infiltration, including in peribronchial, perivascular, and parenchymal regions (B). Contrasting the lack of effect of pretreatment with MPS alone (C), inflammation is suppressed to a similar extent in antigen-challenged OVA-sensitized rabbits that were pretreated either with inhaled budesonide (D) or anti-Gbc peptide (E), whereas pretreatment with gallein has relatively little anti-inflammatory effect (F). Representative photomicrographs (mag. 6100) are from 4 mM sections of H&E stained lung sections. doi:10.1371/journal.pone.0032078.g003 Gbc regulates c-Src-coupled ERK1/2 activation in IL-13- exposed human ASM cells Since signaling initiated by Gi-bc was found to mediate ERK1/ 2-dependent PDE4-induced proasthmatic changes in responsive- ness in b2AR-desensitized rabbit ASM tissues and cultured human ASM (HASM) cells [10,11,23], a series of studies were pursued to systematically evaluate the role of this signaling mechanism in regulating ASM function under the present conditions of ASM sensitization. We initially examined whether HASM cells treated with IL-13 exhibit Gbc-regulated c-Src and ERK1/2 activation, and whether this phenomenon is attributed to direct protein- protein interaction between activated Gbc and c-Src. Accordingly, the effects of Gbc inhibition on IL-13-induced phosphorylation of c-Src protein at residue Tyr416, which denotes the activated autophosphorylated state of the kinase [37], and ERK1/2 were assessed using phospho-c-SrcTyr416- and phospho-ERK1/2-specif- ic antibodies. Corresponding total levels of c-Src and ERK1/2 were also determined in the same immunoblot preparations by stripping the membranes and reincubating with anti-c-Src and - ERK1/2 antibodies. Treatment of HASM cells with IL-13 (50 ng/ml) elicited temporal increases in both c-SrcTyr416 and ERK1/2 phosphorylation that peaked at 10 and 30 min, respectively (Fig. 7A). As demonstrated in Fig. 7B, contrasting the lack of effect of pretreatment with the inert MPS peptide alone (20 mM), peak IL-13-induced phosphorylation of both c-SrcTyr416 and ERK1/2 was suppressed in HASM cells pretreated with maximally effective concentrations of either anti-Gbc blocking peptide (20 mM) or gallein (10 mM). Similar results were also obtained when comparing the effects of IL-13 on c-SrcTyr416 and ERK1/2 phosphorylation in HASM cells at 24 hr following transfection with either an adenovirus vector expressing lacZ (adeno-LacZ), serving as a negative control, or adeno-bARK-ct, PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e32078