Journal: Frontiers in Molecular Neuroscience

Article Title: In vitro methods in autophagy research: Applications in neurodegenerative diseases and mood disorders

doi: 10.3389/fnmol.2023.1168948

Figure Lengend Snippet: In vitro autophagy methods in amyotrophic lateral sclerosis research.

Article Snippet: CMA , Substrate degradation , Hsc70 expression and TDP-43 , Primary lymphomonocytes of sporadic ALS patients, lymphoblastoid cell lines derived from sporadic ALS patients, and SH-SY5Y cells , A : Monitoring the expression of TDP-43 and Hsc70 activity in an ALS model consisting of cells engineered to silence Hsc70 to assess CMA-mediated degradation of TDP-43. D : Peripheral blood mononuclear cells from sporadic ALS patients were used to obtain immortalized lymphoblastoid cells. The expression of Hsc70, LAMP2A, Bag1, Bag3, and LPD-43 was measured in both cell lines to assess CMA and LPD-43 accumulation. The expression of Hsc70 was downregulated in SH-SY5Y cells to assess the participation of Hsc70 in TDP-43 degradation. R : (1) Gene and (2) protein expression assessed by qRT-PCR and Western blot, respectively, targeting Hsc70, LAMP2A, and co-chaperones Bag1 and Bag3. Substrate degradation was evaluated by immunofluorescence analysis targeting TDP-43. (3) Identification of insoluble TDP-43 by filter retardation assay using a Bio-Dot SF microfiltration apparatus and Dot Blot analysis. , .

Techniques: In Vitro, Expressing, Mutagenesis, Biomarker Discovery, Western Blot, Confocal Microscopy, Construct, Immunofluorescence, Ubiquitin Proteomics, Fluorescence, Modification, Immunoprecipitation, Derivative Assay, Activity Assay, Dot Blot