Figure 5. Blockade of α₅β₁ inhibits focal adhesion phosphorylation and impairs IL-13–enhanced tracheal ring contraction.

(A) Representative Western blot and quantitative densitometry for phosphorylated and total vinculin and FAK in human ASM cells treated for 20 minutes with integrin α₅–blocking antibody (P1D6, 10 μg/ml) or mouse IgG control, then plated on poly-l-lysine (300 μg/ml) or poly-l-lysine with either fibronectin (1 μg/ml) or collagen I (1 μg/ml). GAPDH was used as a loading control. *P < 0.05 and **P < 0.01 versus Poly; ^#P < 0.05 versus Poly/FN. n = 3 distinct experiments. GAPDH was used as a loading control. *P < 0.05 and **P <0.01 versus Poly; ^#P < 0.05 versus Poly/FN, by 2-way ANOVA n = 3 distinct experiments. Data represent the mean ± SEM. (B) Contractile force of mouse tracheal rings measured after incubation for 12 hours in DMEM with IL-13 (100 ng/ml) or saline and for 12 hours with α₅-blocking antibody (300 μg/ml) or rat IgG in response to a range of concentrations of the contractile agonist Mch. ***P < 0.001, for IL-13/IgG versus IL-13/anti-α₅. (C) Contractile force of mouse tracheal rings measured after incubation for 12 hours in DMEM with IL-13 (100 ng/ml) or saline, then for 1 hour with a small-molecule inhibitor directed against the α₅β₁ synergy site of fibronectin (ATN-161, 100 μg/ml) or vehicle in response to a range of concentrations of Mch. **P < 0.01 and ***P < 0.001, for IL-13/vehicle versus IL-13/ATN-161. (D) Contractile force of mouse tracheal rings measured after incubation for 12 hours in DMEM with IL-13 (100 ng/ml) or saline (control), then for 1 hour with ATN-161 (100 μg/ml), 20 minutes with rhChy (30 nM), or both, in response to a range of concentrations of Mch. ***P < 0.001, for IL-13/vehicle versus IL-13/ATN-161 plus rhChy. NS, for IL-13/ATN-161 versus IL-13/rhChy versus IL-13/ATN-161 plus rhChy. (B–D) Data represent the mean ± SEM; n = 3–5 rings per group; significance was determined by repeated measures of variance.