实验报告PROTAC-POI体外筛选.PDFVIP

上海美迪西生物医药股份有限公司 上海市浦东新区川大路585 号 邮编：201299 电话：+86 21 实验报告：PROTAC-POI 体外筛选 以某目标蛋白（protein of interest, POI）为例，评价“内源蛋白-EGFP” 293T 稳转株细胞高 通量筛选系统对PROTAC-POI 分子的筛选结果。 首先，利用“POI-EGFP”HEK 293T 细胞稳转株初步检测PROTAC-POI 对细胞中POI 蛋 白水平的下调情况 Fig. 1 Percentage reduction in POI-EGFP fusion protein level HEK 293T cells stably expressing POI-EGFP fusion protein were incubated with PROTAC- POI for 24 h at the concentrations starting from 3333 nM and 1:3 serial dilution. DMSO was used as a solvent control. After lysis of cells, the relative amount of POI-EGFP fusion protein was measured by using Molecular Devices M5 (excitation 488 nm, emission 509 nm). DC50 was determined using the nonlinear regression dose-response equation in GraphPad Prism. 之后，Western Blot 进一步验证PROTAC-POI 对细胞中POI 蛋白水平的下调能力 1 上海美迪西生物医药股份有限公司 上海市浦东新区川大路585 号 邮编：201299 电话：+86 21 Fig. 2 Western Blot Images showing POI degradation by PROTAC-POI A. Images showing POI protein level measured by western blot assay after 24-h treatment with PROTAC-POI at concentrations starting from 3333 nM and 1:3 serial dilution in OCI-LY10 cells. B. Degradation Curve of POI. Relative POI level was quantized using Image J software and normalized to both DMSO and cyclophilin A (a loading control). Then, DC50 was determined using the nonlinear regression dose-response equation in GraphPad Prism. 机制检测 Ⅰ：分析PROTAC-POI 对POI 蛋白水平的下调是否由CRBN E3 Ligase 介导？ Fig. 3 CUL4-CRBN E3 Ligase mediates PROTAC-POI-induced POI Degradation HEK293T cells were pretreated with 25 μM of CRBN inhibitor pomalidomide for 1 h, then incubated with the combination of PROTAC-POI and pomalidomide for another 24 h. Cells were collected and subjected to western blot assay. β-actin was used as a loading control. 机制检测 Ⅱ：分析PROTAC-POI 对POI 蛋白水平的下调是否通过蛋白酶体系统降解途