Western Blotting
BEAS2B cells were stimulated as indicated, subsequently washed with PBS, and lysed in Laemmli buffer [400 mM Tris–HCl, pH 6.8, 20% (v/v) β-mercaptoethanol, 40% (v/v) glycerol, 8% (w/v) SDS, and 0.4% (v/v) bromophenol blue]. After incubation for 10 min at 98°C, equal amounts of lysates were fractionated by 10% polyacrylamide gel (SDS-PAGE) and electrotransferred to Nitrocellulose membranes by a semidry blotting procedure [buffer: 25 mM Tris, 192 mM Glycin, 10% (v/v) methanol; 2.5 mA/cm2 for 1 h 15 min]. Blocking of unspecific binding was performed using 5% BSA solution in 1× TBST [1× TBS, 0.05% (v/v) Tween-20] for at least 1 h. Membranes were stained with antibodies against pY-STAT1 (Tyr701, #9167), STAT1 (#9172), β-Actin (#4970) (Cell Signaling, Leiden, Netherlands; 1:1,000) overnight at 4°C. After three 10 min washing steps in 1 × TBST at room temperature, blots were incubated with secondary antibodies for 1 h at RT [HRP-linked anti-mouse or anti-rabbit (Cell Signaling, Leiden, Netherlands)], followed by additional three 10 min washing steps in 1× TBST at room temperature. Proteins were detected using an enhanced chemiluminescence system (Western lightning™ plus ECL, Perkin-Elmer, Rodgau, Germany). Gels were imaged digitally, and contrast adjustments were applied to all parts of a figure. The prestained molecular weight marker was imaged separately (using transmitted light) and aligned to the digital images of the blots. The ladder is represented on the blots as black bars. Where indicated, membranes were stripped and reprobed. Densitometry was performed using ImageJ software (National Institutes of Health).