SAG (Smoothened Agonist) is a Smo receptor agonist (EC50=3 nM) that is cell-permeable and selective. SAG regulates Smo activity by binding directly to the Smo helix and can activate the Hedgehog signaling pathway.

Smo:3 nM(EC50)

方法：胚胎干细胞 ES 用 SAG (0.5-1.25 µM) 处理 4-6 天，使用 RT-qPCR 基因表达水平。
结果：有丝分裂后 V3 标志物 Sim1 在所有浓度的 SAG 处理组中的表达都增加。用 0.5 μM SAG 处理的组表现出显着更高水平的 Nkx2.2 mRNA 水平。在给定的时间点，Hb9 表达与 SAG 浓度没有显着变化。[1]
方法：非洲绿猴肾脏成纤维细胞样细胞 Cos-1 用 SAG (5-500 nM) 处理，使用 Western Blot 方法检测靶点蛋白表达水平。
结果：SAG 以剂量依赖的方式抑制 Cos-1 细胞中 ER 定位的和 ER 后形式的 Smo-Myc3 与 125I 标记的 PA 环胺的交联。Smo-Myc3 的细胞水平不受激动剂治疗的影响。[2]

方法：为探索 SHH 信号通路是否通过调节线粒体稳态在焦虑中发挥保护作用，将 SAG (10 mg/kg) 腹腔注射给高脂饮食的 C57BL/6 小鼠，每三天一次，持续十二周。
结果：SHH 信号在肥胖中具有神经保护作用，SAG 通过减少线粒体断裂来缓解焦虑样行为。[3]
方法：为研究对发育中的肢体的影响，将 SAG (15-20 mg/kg in lactated Ringer's solution) 单次腹腔注射给妊娠日 (GD) 9.25 的怀孕的 C57BL/6J 小鼠。
结果：SAG 最普遍的作用是对轴前多指畸形的剂量依赖性诱导；缺陷范围从拇指宽到拇指轴前侧两个手指状指的重复。[4]

In vitro Kinase Assays: Kinase assays for CDK1, CDK2 and GSK3-β are all carried out in a radiometric filter binding format. Assays for CDK5 are in DELFIA format and for CDKs 4 and 6 in ELISA format. For CDKs 1 and 2, the relevant CDK and 0.12 μg/mL Histone H1 are incubated in 20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1 mg/mL BSA, 45 μM ATP (0.78 Ci/mmol) and different concentrations of AT7519 for 2 or 4 hours respectively. For GSK3-β, the relevant enzyme and 5 μM glycogen synthase peptide 2 along with 10 mM MOPS pH 7.0, 0.1 mg/mL BSA, 0.001% Brij-35, 0.5% glycerol, 0.2 mM EDTA, 10 mM MgCl2, 0.01% β-mercaptoethanol, 15 μM ATP (2.31 Ci/mmol) and different concentrations of AT7519 are incubated for 3 hours. Assay reactions are stopped by adding an excess of orthophosphoric acid and filtered using Millipore MAPH filter plates. The plates are then washed, scintillant added and radioactivity measured by scintillation counting on a Packard TopCount. For CDK5, CDK5/p35 and 1 μM of a biotinylated Histone H1 peptide (Biotin-PKTPKKAKKL) are incubated in 25 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 0.025% Brij-35, 0.1 mg/mL BSA, 1 mM DTT, 15 μM ATP and different concentrations of AT7519 for 30 minutes. Assay reactions are stopped using EDTA, transferred to Neutravidin-coated plates and phosphorylated peptide quantified by means of a rabbit phospho-cdk1 substrate polyclonal antibody and DELFIA europium-labelled anti-rabbit IgG secondary antibody using time-resolved fluorescence at λex=335 nM, λem=620 nM. For CDK 4 and 6 assays, plates are coated with GST- pRb769-921 and blocked with Superblock. CDK4 or 6 is incubated with 15 mM MgCl2, 50 mM HEPES, pH 7.4, 1 mM DTT, 1 mM EGTA, pH 8.0, 0.02% Triton X-100, 2.5% DMSO and different concentrations of AT7519; the reaction is initiated by addition of ATP. After 30 minutes, reactions are stopped by the addition of 0.5 M EDTA pH 8.0.Plates are then washed and incubated for one hour with the primary antibody (anti- p-Rb Serine 780) diluted in Superblock followed by secondary antibody (alkaline phosphatase linked anti-rabbit) for a further hour. Plates are developed using the Attophos system and fluorescence read on a Spectramax Gemini plate reader at excitation 450 nm and emission 580 nm. In all cases, IC50 values are calculated from replicate curves, using GraphPad Prism software.