Binding assays

Recombinant ROCK-I, ROCK-II, PKN, or citron kinase was expressed in HeLa cells as Myc-tagged proteins by transfection using Lipofectamine, and was precipitated from the cell lysates by the use of 9E10 monoclonal anti-Myc antibody coupled to G protein-Sepharose. Recovered immunocomplexes were incubated with various concentrations of [32P] ATP and 10 mg of histone type 2 as substrates in the absence or presence of various concentrations of either Y-27632 at 30°C for 30 min in a total volume of 30 mL of the kinase buffer (50 mM HEPES-NaOH, pH 7.4, 10 mM MgCl₂, 5 mM MnCl₂, 0.02% Briji 35, and 2 mM dithiothreitol). PKCa was incubated with 5 mM [32P] ATP and 200 mg/mL histone type 2 as substrates in the absence or presence of various concentrations of either Y-27632at 30°C for 10 min in a kinase buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM CaCl₂, 5 mM magnesium acetate, 25 mg/mL phosphatidyl serine, 50 ng/mL 12-O-tetradecanoylphorbol-13-acetate and 0.001% leupeptin) in a total volume of 30 mL. Incubation was terminated by 43 Laemmli sample buffer (10 mL).

Cell lines

HeLa cells, Swiss 3T3 cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37°C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20°C for several months.

Reacting condition

0.3 and 1 μM, 30 min or 24 h

Applications

Y-27632 inhibited the kinase activity of both ROCK-I and ROCK-II in vitro, and this inhibition was reversed by ATP in a competitive manner. Y-27632 abolished stress fibers in Swiss 3T3 cells at 10 μM, but the G1-S phase transition of the cell cycle and cytokinesis were little affected at this concentration. In Swiss 3T3 cells, prior treatment of cells with Y-27632 for 30 min prevented cell shape and actin-stress fibers changes in a concentration-dependent manner; almost complete inhibition was observed at 10 μM. 10 and 100 μM Y-27632 prolonged the lag time and delayed the appearance of BrdU-labeled cells in a concentration-dependent manner, delays of about 1 and 4 h. In HeLa cells, 30 μM Y-27632 inhibited cytokinesis.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Ishizaki T, Uehata M, Tamechika I, et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases[J]. Molecular pharmacology, 2000, 57(5): 976-983.