Y-27632 dihydrochloride (ROCK抑制剂) (RPM0007)

体外活性/In vitro:
1.用Y-27632（5-20 μM）处理marmoset iPSC 7天，使用 AKP 检测克隆形成情况。Y-27632显著提高marmoset iPSC的克隆效率。[1]
2.用Y-27632（5 μmol/L）处理成人脂肪组织衍生干细胞ADSCs 1 h，检测细胞形态变化。Y-27632剂量依赖性诱导ADSCs的神经元分化，ADSCs的神经元样细胞百分比为（93.5±4.7）%。[2]
3.用Y-27632（1-10 μM）处理食蟹猴胚胎干细胞cyES 24 h，使用Flow Cytometry方法进行活-死染色，检测BrdU。Y-27632促进cyES存活细胞增加。Y-27632没有促进细胞增殖，但保护细胞在单细胞消化后免于细胞死亡。[3]

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].

1.产品描述/Description：Y-27632二盐酸盐是ATP竞争性的ROCK-I和ROCK-II抑制剂。|2.中文名称/ChineseName：Y-27632二盐酸盐。|3.别称/Synonyms：反式-4-[(R)-1-氨基乙基]-N-(4-吡啶基)环己烷甲酰胺二盐酸盐。|4.CAS号/CASNO.：129830-38-2。|5.化学式/Formula：C14H21N3O·2HCl。|6.分子量/M.Wt：320.26|7.外观/Appearance：固体|8.保存条件/Storage：Powder：-20℃，3年,-80℃，1年。收到重组蛋白产品之后请检查蛋白冻干粉末是否贴于瓶底，如果粉末浮起，开盖之前请先低温离心。将蛋白用说明书中指定的缓冲液复溶至0.1-0.5 mg/mL（请注意蛋白复溶浓度不能低于0.1 mg/mL），室温平衡5-10 min保证充分溶解，复溶过程中请不要剧烈涡旋及吹打蛋白溶液。如需长期储存，建议复溶时添加载体蛋白或者稳定剂（如0.1% BSA, 5% HSA, 10% FBS 或者 5% 海藻糖），同时将复溶后的蛋白溶液按照需求进行分装，储存于-20°C至-80°C，随取随用，避免反复冻融。