Cyclosporin A is a naturally occurring cyclic polypeptide that is the active metabolite of a fungus. Cyclosporin A is an immunosuppressant that binds to procyclins and inhibits calcineurin (IC50=7 nM).

Calcineurin phosphatase:7 nM

方法：胶质瘤细胞 C6 用 Cyclosporin A (10 μM) 在缺氧条件下处理 4 h，使用 Western Blot 方法检测靶点蛋白表达水平。
结果：4 h 缺氧诱导了内源性 HIF-1α 蛋白的大量积累。Cyclosporin A 阻止了缺氧诱导的 HIF-1α 的大部分稳定。[1]
方法：人结肠癌细胞 CACO-2 用 Cyclosporin A (2 μM) 处理 24-72 h，使用 Flow Cytometry 检测细胞周期情况。
结果：在用 Cyclosporin A 处理后的 G0/G1 阶段检测到细胞的积聚。[2]

方法：为检测体内对肌肉疾病的活性，将 Cyclosporin A (5 mg/kg in olive oil) 腹腔注射给肌病 Col6a1-/- 小鼠，每天两次，持续十天。
结果：Cyclosporin A 影响 Col6a1-/- 小鼠的卫星细胞活性并触发再生纤维的形成。[3]
方法：为检测对缺血再灌注损伤 (IRI) 的作用，将 Cyclosporin A (3 mg/kg，在缺血前 1 h 或 10 min；10 mg/kg，在缺血前 10 min) 腹腔注射给缺血再灌注的 C57BL/6J 小鼠。
结果：Cyclosporin A 10 mg/kg-10 min 和 Cyclosporin A 3 mg/kg-1 h 组的死亡率和肾功能显著高于 Cyclosporin A 3 mg/kg-10 min 组。[4]

Phosphatase Assay: Purified bovine brain calcineurin and calmodulin are purchased. Reaction mixtures with purified enzyme contains 100 nM calcineurin, 100 nM calmodulin, and 5 μM 32P-labeled phosphopeptide, in 60 μl (total volume) of assay buffer containing 20 mM Tris (pH 8), 100 mM NaCI, 6 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mg of bovine serum albumin per ml, and either 0.1 mM CaCl2 or 5 mM EGTA. Reaction mixtures with cell lysates contains 20 μl of undiluted lysate, 5 μM 32P-labeled phosphopeptide, and 40 μl of assay buffer. Where indicated, reaction mixtures contains 50 μM peptide 412 or 413 and/or 500 nM okadaic acid, a specific inhibitor of phosphatases 1 and 2A; 500 nM okadaic acid is sufficient for inhibition of Ca2+-independent phosphatases, whereas higher concentrations partially inhibit Ca2+-dependent activity as well. After 15 min at 30°C, reactions are terminated by the addition of 0.5 ml of 100 mM potassium phosphate buffer (pH 7.0) containing 5% trichloroacetic acid. Free inorganic phosphate is isolated by Dowex cation-exchange chromatography and quantitated by scintillation counting as described.

Immunosuppressive agents are dissolved in ethanol at concentrations 1000-fold more than the concentration desired for cell treatments. Cells (106) are suspended in 1 ml of complete medium in microcentrifuge tubes; 1 μl of ethanol or of the ethanolic solution of Cyclosporin A is added, and the cells are incubated at 37°C for 1 hr. Cells are washed twice with 1 ml of PBS on ice and lysed in 50μl of hypotonic buffer containing 50 mM Tris (pH 7.5); 0.1 mM EGTA; 1 mM EDTA; 0.5 mM dithiothreitol; and 50 μg of phenylmethylsulfonyl fluoride, 50 μg of soybean trypsin inhibitor, 5 μg of leupeptin, and 5 μg of aprotinin per ml. Lysates are subjected to three cycles of freezing in liquid nitrogen followed by thawing at 30°C and then are centrifuged at 4°C for 10 min at 12,000×g.(Only for Reference)