Protocols to monitor TFEB activation following lysosomal damage in cultured cells using microscopy and immunoblotting

Abstract

Summary: 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).

Keywords

• Antibody
• Cell Biology
• Cell culture
• Cell separation/fractionation
• Cell-based Assays
• Microscopy