Protocol Archive

This 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.

The 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.

The authors describe performing co-immunoprecipitation experiments in HEK293 cells using GFP-trap beads.

This 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).

Ectopic 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.

This 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.

The authors describe HEK293 cell culture for the purpose of co-immunoprecipitation experiments.

The 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.

The 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.

Here, 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."

Three standards of the field for determining phase separation are: (A) particles undergo fission and fusion like droplets of water, (B) individual puncta recover fluorescence after photobleaching, and (C) particles dissipate upon exposure to 1,6-Hexanediol. This protocol describes the authors’ approach to investigating standards B and C.

The live imaging of NEMO occupancy on damaged mitochondria was a necessary complement to the authors’ parallel fixation studies. Their reporting of these results would not have been possible without real-time, live cell imaging.

The authors describe procedure and equipment used for live-imaging of synaptic vesicle precursors. This was performed using DIV21 human iPSC-derived excitatory glutamatergic neurons. Equipment and software used varied based on scheduled upgrades to microscopy equipment during the course of this study.

This protocol describes how to perform microscopy-based bead protein-protein interaction assay with GST- or mCherry-tagged proteins as baits and fluorescently-tagged proteins as preys. The protocol requires to have purified proteins and allows to monitor protein-protein interaction in an equilibrium state. The fluorescent signal can be quantified.

This protocol describes the creation of YIPF4 knockout cell lines in H9 hESC cells using CRISPR-Cas9. Associated with preprint: https://doi.org/10.1101/2022.12.06.519342

Associated with preprint: https://doi.org/10.1101/2023.06.26.546565

Associated with preprint: https://doi.org/10.1101/2023.06.26.546565

Associated with preprint: https://doi.org/10.1101/2023.06.26.546565

This protocol describes the preparation and culture of mouse primary cortical neurons for live-imaging experiments. Neurons were transfected 16-24 hours before imaging using Lipofectamine 2000.

The authors developed this protocol to test levels of NF-kB response genes in a cell model transiently over-expressing Parkin. With this technique the authors found significant upregulation of key pro-inflammatory genes normalized to a housekeeping gene, Gapdh.