Measurement of myosin regulatory light chain phosphorylation.

Smooth muscle myosin regulatory light chain (RLC) phosphorylation was analyzed as previously described (28, 47). Tracheal muscle tissue strips attached to force transducers were rapidly frozen using liquid nitrogen-cooled tongs. Proteins were extracted in 8 M urea, 20 mM Tris base, 22 mM glycine, and 10 mM dithiothreitol. Unphosphorylated and phosphorylated myosin RLCs were separated by urea/glycerol-PAGE, transferred to nitrocellulose, and immunoblotted for myosin RLC. The proportions of phosphorylated and unphosphorylated myosin RLCs were quantified by scanning densitometry, and myosin RLC phosphorylation was calculated as the ratio of phosphorylated RLC to total RLC.