Fudan University Shanghai Cancer Center, Shanghai Key Laboratory of Medical Epigenetics, Shanghai Key Laboratory of Radiation Oncology, Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
Fudan University Shanghai Cancer Center, Shanghai Key Laboratory of Medical Epigenetics, Shanghai Key Laboratory of Radiation Oncology, Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
STAR Protoc. 2023 Dec 15;4(4):102640. doi: 10.1016/j.xpro.2023.102640. Epub 2023 Oct 12.
While several inhibitors targeting RNA polymerase II (Pol II) kinases have been applied for inhibiting RNA Pol II phosphorylation, there are few approaches for inducing RNA Pol II hyperphosphorylation. Here, we present a protocol for constructing the INTS8 degradation tag (dTAG) system combined with ectopic expression of N-terminally truncated INTS8 (INTS8-ΔN) in DLD-1 cells. We describe steps for INTS8-dTAG cell line construction, validation of knockin and degradation, and INTS8-ΔN rescue. We then detail validation of RNA Pol II phosphorylation upregulation. For complete details on the use and execution of this protocol, please refer to Hu et al. (2023)..
虽然已经有几种针对 RNA 聚合酶 II (Pol II) 激酶的抑制剂被用于抑制 RNA Pol II 的磷酸化，但诱导 RNA Pol II 过度磷酸化的方法却很少。在这里，我们介绍了一种构建 INTS8 降解标签 (dTAG) 系统的方案，该系统结合了在 DLD-1 细胞中外源性表达 N 端截断的 INTS8 (INTS8-ΔN)。我们描述了 INTS8-dTAG 细胞系构建、敲入和降解验证以及 INTS8-ΔN 挽救的步骤。然后，我们详细说明了 RNA Pol II 磷酸化上调的验证。有关该方案使用和执行的完整详细信息，请参阅 Hu 等人。(2023)。
