Fig 3. Gβγ-regulated c-Src and ERK1/2 activation is constitutively increased in asthmatic HASM cells and evoked in normal HASM cells stimulated with IL-13.

(A) Western blot analysis demonstrating that, relative to normal cells, untreated asthmatic HASM cells exhibit constitutively increased free Gβ levels and correspondingly reduced co-localization of Gβ with immunoprecipitated Gα, denoting an intrinsic state of G protein activation. This pattern of Gβ distribution is acutely reversed in asthmatic HASM cells treated either with gallein or the anti-Gβγ blocking peptide (1 μM; x 30 min), By comparison, neither Gβγ inhibitor alters Gβ distribution in normal cells. (B) Immunoblots comparing 3 normal (N1–N3) and 3 asthmatic (A1–A3) HASM cell lines demonstrate that, under the same protein loading conditions yielding similar β-actin levels, constitutive expression of phosphorylated c-Src (p-Src) together with free Gβ protein are distinctly increased in the asthmatic HASM cell lines. Representative immunoblots in (C) and (D) demonstrate that, relative to normal cells, constitutively increased p-Src and p-ERK1/2 levels detected in asthmatic HASM cells, respectively, are acutely suppressed by treatment with either the anti-Gβγ blocking peptide or gallein (1 μM x 30 min), similar to the suppressive effect of treatment with the c-Src-selective inhibitor, SU6656. (E) Treatment of normal HASM cells with IL-13 (50 ng/ml) acutely evokes temporal increases in free Gβ protein levels, peaking at 60–90 min and remaining elevated above baseline at 120 min. (F) Co-IP experiment demonstrating that, relative to unstimulated cells, IL-13-treated (50 ng/ml x 60 min) normal HASM cells exhibit increased co-localization of p-Src with immunoprecipitated Gβ, and the latter is abrogated in IL-13-exposed HASM cells pretreated with either anti-Gβγ blocking peptide or gallein. The immunoblots shown in A-F are representative of 3–4 experiments.