Fecal metagenomic sequencing data for PD patients and controls from the BioCollective
By savannah onFecal metagenomic sequencing data associated with Boktor et al., 2023. This dataset includes samples from the BioCollective cohort.
Macros and pipelines for analysis of image analysis
By savannah onImageJ macros and pipelines for analysis of pUb puncta, Keima Ratio, p62nuclei, and Parkin recruitment to mitochondria.
Proteomics of iNeurons or HeLa cells with or without FBXO7
By savannah onTMT proteomics datasets from FBXO7-/- hESC differentiation to iNeurons and HeLa cells.
Proteostasis and lysosomal quality control deficits in Alzheimer’s disease neurons
By savannah onCompounds enhancing lysosomal function broadly ameliorate AD-associated pathologies. The authors’ findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis.
Raw tomogram of a TauRD-YFP aggregate in a TauRD-YFP expressing HEK293T cells and primary mouse neurons
By savannah onRaw tomogram of a TauRD-YFP aggregate in a TauRD-YFP expressing Hek293T cells. Accession numbers: HEK293T: https://www.ebi.ac.uk/emdb/EMD-13739 Primary mouse neurons: https://www.ebi.ac.uk/emdb/EMD-13740
Scripts for analysis of total proteomes in response to nutrient stress
By savannah onScripts for analysis of total proteomes in response to nutrient stress. Associated with preprint: https://doi.org/10.1101/2022.12.06.519342
Web portal (shiney ap) for analysis of total proteomes in response to nutrient stress
By savannah onWeb portal (shiney app) for analysis of total proteomes in response to nutrient stress. Associated with preprint: https://doi.org/10.1101/2022.12.06.519342
Astrocyte isolation and culturing
By savannah onThis protocol details steps for preparing an astrocyte monoculture from mouse pups. From dissection to plated samples ready to be transfected, treated, lysed, or imaged, the protocol takes 20-25 days.
Cell lysis and gel electrophoresis for protein analysis of HeLa cells
By savannah onThe authors present multiple protocols used for biochemical analysis of protein expression and association. The authors’ studies demonstrated that NEMO recruitment to damaged mitochondria occurs in specific circumstances, and NEMO colocalization with p62 is also dependent on multiple factors.
Co-immunoprecipitation using GFP-trap
By savannah onThe authors describe performing co-immunoprecipitation experiments in HEK293 cells using GFP-trap beads.
Expression and purification of human NEMO (GST-GFP-NEMO)
By savannah onThis protocol describes how to express and purify human NEMO (IKK-γ) tagged N-terminally with GST and EGFP. The expression is performed with E. coli Rosetta pLysS cells. The protein is purified via a GST batch purification and gel filtration (SEC).
Fixation and imaging of HeLa cells after mitochondrial depolarization
By savannah onEctopic expression of p62/SQSTM1 often induces puncta formation and poor cell health due to p62’s proclivity to multimerize and form filaments. We noted modifying fixation protocol to visualize various endogenous ATG8 proteins, which are difficult to over-express in our system. Fixation techniques are a critical complement to studies in live cells.
Fixing hippo neurons to assess endogenous NEMO during oxidative stress
By savannah onThis protocol details how to assess endogenous NEMO during oxidative stress in fixed hippocampal rat neurons. Mitochondrial damage was induced by Antimycin A, endogenous NEMO was visualized with the commercially available anti-NEMO primary antibody (abcam), and mitochondria were visualized by a mito-targeted construct, Mito-SNAP.
HEK293 cell culture for co-immunoprecipitation experiments
By savannah onThe authors describe HEK293 cell culture for the purpose of co-immunoprecipitation experiments.
HeLa culture, transfection, and labeling of Halo-fusion proteins
By savannah onThe authors developed a protocol to investigate the role of the NEMO in Parkin-dependent mitochondrial clearance. Halo-fusion constructs, including NEMO and OPTN used in the study, allowed the authors to visualize the exogenously expressed proteins conjugated to chemical ligands in a variety of colors. This and the accompanying protocols were critical to the characterization of NEMO’s involvement in mitophagy.
Image processing to investigate NEMO recruitment and involvement in mitophagy and inflammatory signaling
By savannah onThe authors approached image analysis in a multitude of creative, effective ways, and importantly the authors maintained consistency of analysis within experiments in order to present the results with integrity and reproducibility.
Immunostaining of iPSC-derived neurons for quantification of synaptic proteins
By savannah onHere, we fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal proteins of interest. For preceding culture of neurons, see "Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes."