Cell treatment with antagonists

Cultured anal sphincter smooth muscle cells were maintained under controlled conditions. To investigate the role of muscarinic acetylcholine receptors (mAChRs), cells were treated with selective antagonists: atropine (A0132, Sigma-Aldrich,USA) at a concentration of 10 μM for non-specific mAChR blockade, 4-DAMP (0929, Tocris Bioscience, USA) at a concentration of 1 μM for M3 mAChR inhibition, and AF-DX 116 (0674, Tocris Bioscience, USA) at a concentration of 2 μM for M2 mAChR inhibition. All incubation lasted for 1h. Following treatment, cellular responses were evaluated through Western blotting.