Lysosomal Trafficking of Neurodegenerative Proteins

To confirm trafficking of WT and mutant α-syn, TDP-43 and tau into the lysosomes of our SH-SY5Y cells, we performed a series of lysosome isolation experiments. SH-SY5Y cells were differentiated and treated with MG132 as previously described [37 (link), 38 (link)]. Cells were harvested on day 14 post-differentiation and the lysosomes of these cells isolated using a gradient-ultracentrifuge based technique (Lysosome Enrichment Kit for Tissues and Cultured Cells from Thermofisher, # 89,839). Successful isolation and enrichment of lysosomes were confirmed by Western Blot using a Lamp1 antibody (Cell Signaling #9091).

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Sampognaro P.J., Arya S., Knudsen G.M., Gunderson E.L., Sandoval-Perez A., Hodul M., Bowles K., Craik C.S., Jacobson M.P, & Kao A.W. (2023). Mutations in α-synuclein, TDP-43 and tau prolong protein half-life through diminished degradation by lysosomal proteases. Molecular Neurodegeneration, 18, 29.

Publication 2023

Lysosome Enrichment Kit for Tissues and Cultured Cells Lamp1 antibody

Antibody Cultured cells Isolation Lamp1 Lysosomes Mg132 Tdp 43 Tissues Western blot

Corresponding Organization :

Other organizations : University Memory and Aging Center, University of California, San Francisco, Coherus BioSciences (United States), Icahn School of Medicine at Mount Sinai

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Variable analysis

independent variables

Treatment with MG132

dependent variables

Trafficking of wild-type and mutant α-syn, TDP-43, and tau into the lysosomes

control variables

SH-SY5Y cells were differentiated
Cells were harvested on day 14 post-differentiation
Lysosome isolation using a gradient-ultracentrifuge based technique

controls

Positive control: Lamp1 antibody used to confirm successful isolation and enrichment of lysosomes

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