Constitutive activation of an epithelial signal transducer and activator of transcription (STAT) pathway in asthma
J. Clin. Invest. Deepak Sampath, et al. 103:1353 doi:10.1172/JCI6130 [Go to this article.]

Figure 3
Stat1 activation in asthma detected by protein phosphorylation. (a) Immunoprecipitation/immunoblot results for phosphorylated Stat1 from 3 control (lanes 1–3), 3 asthmatic (lanes 4–6), and 2 chronic bronchitis (lanes 7 and 8) subjects, as well as primary-culture airway epithelial cells stimulated with IFN-γ (100 U/mL for 24 hours; lane 9). In each case, Stat1 was immunoprecipitated from epithelial cell lysates and subjected to immunoblotting with anti-phosphotyrosine mAb conjugated to horseradish peroxidase followed by enhanced chemiluminescence. Arrows indicate positions of a nonspecific band (NB) and phosphorylated Stat1. (b) Results for total Stat1 after membranes were reprobed with anti-Stat1 mAb. Arrows indicate position of Stat1. (c) Ratio of phosphorylated Stat1 to total Stat1 band intensity for each condition, based on densitometry of immunoblots in a and b. Using the same techniques, d and e show comparison of airway epithelial (E) and bronchoalveolar macrophage (M) levels of Stat1 activation in 2 control (lanes 1–4) and 2 asthmatic (lanes 5–8) subjects. Titration of sample amounts to provide equal levels of Stat1 in each lane still showed no evidence of phosphorylation in samples from nonasthmatic subjects (not shown). (f and g) Immunoblots (without prior immunoprecipitation) of airway epithelial cell lysates for IRF-1 (f), Stat1 (g) under conditions that separate Stat1α from Stat1β, and control β-actin (h).