体外活性
体外活性/In vitro:
1.用SB431542（2-10 μM）和TGF-β1（12.5 ng/mL）处理人肾细胞293T、人类结肠癌来源的非致瘤性细胞FET和人肺腺癌细胞A549 1 h，Western Blot检测靶点蛋白表达水平。TGF-β1诱导了Smad2/3和Smad4之间的复合物形成。SB431542以剂量依赖的方式，通过阻断TGF-β诱导的磷酸化和Smad2/3的激活，阻断复合物形成。[1]
2.用SB431542（1-10 μM）处理人胶质母细胞瘤细胞D54MG 24 h，Transwell检测细胞迁移情况。SB431542抑制TGF-βRI信号传导以浓度依赖的方式阻断D54MG细胞的细胞迁移。[2]
3.用含SB431542（10 nM）和Noggin（500 ng/mL）的KSR培养基培养人多能干细胞hESCs 11天，免疫荧光检测细胞分化情况。与单独使用Noggin或SB431542时不到10%的PAX6+细胞相比，用Noggin和SB431542联合处理显著提高了神经诱导的效率，使其超过总细胞的80%。[3]
体内活性/In vivo:
1.将SB431542（10 mg/kg in 20% DMSO/80% corn oil）腹腔注射给携带小鼠乳腺癌肿瘤4T1的Balb/c小鼠，每周三次，持续四周，检测体内抗肿瘤活性。SB431542可显著抑制4T1乳腺肿瘤的肺转移。[4]
2.将SB431542（10 mg/kg）腹腔注射给大面积肩袖撕裂的C57B/6J小鼠模型，每天一次，持续二或六周，研究肌腱损伤的治疗方法。SB431542抑制TGF-β1信号传导可减少纤维化、脂肪浸润和肌肉重量减轻。SB431542治疗通过促进纤维/脂肪生成祖细胞（FAP）的凋亡，减少了损伤肌肉中FAP的数量。[5]
激酶实验/Kinase Assay:
Kinase assays were performed with 65 nM GSTALK5 and 184 nM GST-Smad3 in 50 mM HEPES, 5 mM MgCl2, 1 mM CaCl2, 1 mM dithiothreitol, and 3 M ATP. Reactions were incubated with 0.5 μCi of [33P]γATP for 3 h at 30°C. Phosphorylated protein was captured on P-81 paper, washed with 0.5% phosphoric acid, and counted by liquid scintillation. Alternatively, Smad3 or Smad1 protein was also coated onto FlashPlate Sterile Basic Microplates. Kinase assays were then performed in FlashPlates with same assay conditions using either the kinase domain of ALK5 with Smad3 as a substrate or the kinase domain of ALK6 (BMP receptor) with Smad1 as substrate. Plates were washed three times with phosphate buffer and counted by TopCount [2].
