previously (28 (link)). Briefly, BSM strips
removed from silicone elastomer were suspended in organ baths containing Tyrode
buffer and stretched to generate a force of 1.5 g. Following equilibration, the
strips were stimulated with either high potassium (120 mM KCl) or carbachol (10
μM) and then frozen rapidly by clamp-freezing in liquid nitrogen,
followed by immersion in a dry ice/acetone slurry containing 10% trichloroacetic
acid (TCA) and 10 mM DTT. The strips were slowly thawed at room temperature,
washed in acetone, air dried, and then subjected to homogenization and
precipitated the protein in 10% TCA with 10 mM DTT. The samples were centrifuged
at 12,000 rpm for 10 min and the protein pellet was washed with diethylehter,
air-dried and resuspended in sample buffer. Equal amounts of proteins were
resolved on SDS-PAGE and transferred to polyvinylidene difluoride membranes
(Millipore, Bedford, MA). The proteins were subjected to immunoblot analysis
using phospho-myosin light chain (Thr18/Ser19) (Cell Signaling Technology,
Danvers, MA, USA) and myosin light chain antibodies (Abcam, Cambridge, MA,
USA).