Store at -20℃. This product is stable at -20℃ up to 3 years from the date of receipt . After reconstitution, the solution is stable at -20℃ for 6 months, at -80℃ for up to 1 year.未开盖的干粉蛋白在 -20°C至-80°C可保存12个月； 复溶之后，蛋白溶液在-20°C及以下可保存3个月，在2-8℃可保存1周。
体内活性/In vivo: 1.将Y-27632（2 or 30 mg/kg in drinking water）口服给ALS模型的SOD1G93A小鼠，持续137天，研究Y-27632在运动神经元疾病的治疗潜力。Y-27632 2 mg/kg治疗的效果不佳，30 mg/kg治疗可改善雄性小鼠的运动功能，雌性小鼠仅表现出有限的改善。[4] 2.将Y-27632（30 mg/kg）口服给药给DMN诱导肝纤维化的大鼠，每天一次，持续四周，研究Y-27632对肝纤维化的影响。Y-27632治疗显著减少了DMN诱导的肝纤维化的发生，并降低了肝脏中胶原和羟脯氨酸的含量以及α-SMA的表达。[5]
激酶实验/Kinase Assay: Recombinant ROCK1/2, PKN, or citron kinase is expressed in HeLa cells as Myc-tagged proteins by transfection using Lipofectamine and is precipitated from the cell lysates by the use of 9E10 monoclonal anti-Myc antibody coupled to G protein-Sepharose. Recovered immunocomplexes are 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 or Y-30141 at 30°C for 30 min in a total volume of 30 μL of the kinase buffer containing 50 mM HEPES-NaOH, pH7.4, 10 mM MgCl2, 5 mM MnCl2, 0.02% Briji 35, and 2 mM dithiothreitol. PKCa is incubated with 5 μM [32P]ATP and 200 μg/mL histone type 2 as substrates in the absence or presence of various concentrations of either Y-27632 or Y-30141 at 30°C for 10 min in a kinase buffer containing 50 mM Tris-HCl, pH 7.5, 0.5 mM CaCl2, 5 mM magnesium acetate, 25 μg/mL phosphatidylserine, 50 ng/mL 12-O-tetradecanoyl phorbol-13-acetate and 0.001% leupeptin in a total volume of 30 μL. Incubation is terminated by the addition of 10 μL of 43 Laemmli sample buffer. After boiling for 5 min, the mixture is subjected to SDS-polyacrylamide gel electrophoresis on a 16% gel. The gel is stained with Coomassie Brilliant Blue and then dried. The bands corresponding to histone type 2 are excised, and the radioactivity is measured [1].
细胞实验/Cell Research: HeLa cells are plated at a density of 3×104 cells per 3.5-cm dish. The cells are cultured in DMEM containing 10% FBS in the presence of 10 mM Thymidine for 16 h. After the cells are washed with DMEM containing 10% FBS, they are cultured for an additional 8 h, and then 40 ng/mL of Nocodazole is added. After 11.5 h of the Nocodazole treatment, various concentrations of Y-27632 (0-300 μM) or vehicle is added and the cells are incubated for another 30 min [1].
