Methods for Studying TNFα-Induced Autophagy
Autophagy is an evolutionarily conserved cellular mechanism in eukaryotes that plays an important role in the maintenance of cellular homeostasis. The autophagy process maintains protein homeostasis by recycling damaged organelles and degrading many long-lived proteins in conjunction with the ubiquitin-proteasome system. Cytokines are low-molecular-weight secreted proteins that regulate a broad range of biological activities. For instance, pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα) induce inflammation, autophagy, and apoptotic cell death. In this chapter, we discuss experimental techniques such as immunoblotting and fluorescence microscopy that can be utilized to measure autophagy in response to TNFα treatment.
Key wordsApoptosis Autophagy Bafilomycin A1 Chloroquine Fluorescence microscopy Immunoblotting LC3 p62 TNFα
Research reported in this chapter was supported by an award from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC2GM125550 to VVD and by the College of Pharmacy and Health Sciences, St. John’s University startup fund to VVD.
- 20.He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43:67–93. https://doi.org/10.1146/annurev-genet-102808-114910CrossRefPubMedPubMedCentralGoogle Scholar