R blebbistatin

4-aminopyridine (4-AP) and pyruvate were purchased from Sigma (St. Louis, MO, USA). Hexahydro-1-[(5-iodo-1-naphthalenyl) sulfonyl]-1H-1,4-diazepine hydrochloride (ML-7) and (S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1152) were purchased from Calbiochem (San Diego, CA, USA). Ethyleneglycol-bis (β-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) was purchased from Dojin (Kumamoto, Japan). Tetrodotoxin (TTX) was purchased from Wako (Osaka, Japan). D-(−)-2-amino-5-phosphonopentanoic acid (APV), bicuculline, (S)-(−)-blebbistatin, (R)-(+)-blebbistatin, thapsigargin and nifedipine were purchased from Tocris (Ellisville, MO, USA).

Recombinant TAT-C3 was prepared as previously described30 (link), and used at a concentration of 50 μg/ml. TAT-C3 is a cell permeable form of exoenzyme C3 transferase (from Clostridium botulinum), a Rho-specific inhibitor. It consists of C3 transferase fused to the protein transduction domain TAT (trans-activator of transcription), encoded by the human immunodeficiency virus gene Tat31 (link). Y-27632 (Sigma-Aldrich, Germany), dissolved in DMSO to give a 30 mM stock solution, was used at 30 μM. Racemic RS-blebbistatin, R-(+)-blebbistatin and S-(−)-blebbistatin were obtained from Tocris Bioscience (Bristol, United Kingdom) and dissolved in anhydrous dimethyl sulfoxide (DMSO) to yield 50 mM stock solutions.