Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
Department of Genetics, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
STAR Protoc. 2022 Jan 7;3(1):101018. doi: 10.1016/j.xpro.2021.101018. eCollection 2022 Mar 18.
Following lysosomal damage, activation and nuclear translocation of transcription factor EB (TFEB) is the key event to maintain lysosomal homeostasis. Here, we describe steps to induce lysosomal damage in HeLa cells. This can be followed by monitoring the changes in TFEB localization using widefield fluorescence microscopy. As a complementary approach, we describe the use of immunoblotting to follow the activation and localization of TFEB in cell lysates. These protocols enable quantitative analysis of TFEB. For complete details on the use and execution of this protocol, please refer to Nakamura et al. (2020).
在溶酶体受到损伤后，转录因子 EB（TFEB）的激活和核转位是维持溶酶体稳态的关键事件。在这里，我们描述了诱导 HeLa 细胞溶酶体损伤的步骤。随后，可以使用宽场荧光显微镜监测 TFEB 定位的变化。作为一种补充方法，我们描述了使用免疫印迹来跟踪细胞裂解物中 TFEB 的激活和定位。这些方案可实现 TFEB 的定量分析。有关此方案的使用和执行的完整详细信息，请参阅 Nakamura 等人（2020 年）。
