11/21/2023 0 Comments Chaperone mediated autophagy assays![]() ![]() To determine the flux, we used another set of samples treated with E64D, (L-trans-Epoxy-succinyl-leucylamido(4-guanidino)butane), a cathepsin inhibitor, which would serve as controls. By measuring the cleavage, we can measure the levels of CMA activity. ![]() The cleavage of KFERQ-AMC due to lysosomal hydrolysis causes free AMC to release which is excited at 355 nm and fluoresces at 460 nm. AMC when attached to the substrate remains in the quenched state and does not fluoresce. We then aim to demonstrate the use of KFERQ-AMC (Lys- Phe-Asp-Arg-Gln-AMC) fluorogenic substrate to measure CMA activity. We first extract intact lysosomes from adult mouse heart, liver, and kidney and cultured mouse embryonic fibroblasts. In this project, we have established a fluorescent-based CMA activity assay to measure CMA in cells and tissues. LAMP2A protein expression has been used previously to assess CMA function. CMA uses protein type 2a (LAMP2A) receptors, which are specific to CMA, to target proteins that contain a KFERQ-like motif for lysosomal degradation. Chaperone-mediated autophagy (CMA) is a protein degradation pathway unique to mammalian cells. Misfolded and damaged proteins are usually checked by protein degradation pathways which are essential to maintain proteostasis. College of Health 38 Development of a Novel Fluorogenic-Based Assay to Measure Chaperone Mediated Autophagy in Cells and TissuesĪnila Jonnavithula Rajeshwary Ghosh Megan Tandar Scott N.Orton MacKenzie Woodrum Sihem Boudina and J David Symons (Nutrition & Integrative Physiology)įaculty Mentor: Rajeshwary Ghosh (Nutrition & Integrative Physiology, University of Utah) ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |