Protocol Archive

Protocol for the biochemical purification and analysis of SKP1-FBXO7 complexes.

This protocol delineates a qPCR method to confirm the expression of circRNAs extracted from brain samples.

This protocol details Halo-LC3B processing assay to assess autophagy.

This protocol details the generation of stable ATG3-mCherry wild-type or mutant HeLa cells with HaloTag-LC3B using lentivirus.

This protocol details generation of ATG3 KO Hela cells stably expressing HaloTag-LC3B.

Cloning of ATG3 construct for production of recombinant (GFP tagged) ATG3 variants.

LC3 lipidation reaction assay on liposomes (SUVs, All Unilamellar vesicles).

LC3 lipidation reaction assay on Giant Unilamellar vesicles (GUVs).

This protocol describes how to express and purify human SINTBAD tagged C-terminally with eGFP.

This protocol describes how to express and purify human NAP1 tagged C-terminally with mCherry.

This protocol describes in vitro kinase assay, in which PI3Kc is tested for phosphorylation by TBK1 and ULK1 complex either by western blotting (S29 on ATG14L subunit) or mass spectrometry analysis.

The immunoprecipitation (IP) of FLAG-tagged LRRK2 from whole cell lysates to assess its interaction with Rab12. This method can be used to screen the impact that LRRK2 mutations have on its binding to Rab12 in cells, as well as the effect of any compound or cell treatment on LRRK2 interaction with Rab12.

This is a collection of protocols for the manuscript "Synaptotagmin-1-dependent phasic axonal dopamine release is dispensable for basic motor behaviors in mice."

This protocol described the production of human cortical neurons from induced pluripotent stem cells in cocultured with commercially available rat astrocytes.

The authors adapted a previously described method (Pantazis et al., 2022) for employing Piggybac transfection to stably express doxycycline-inducible NGN2 in human iPSCs. After stable integration of NGN2, proceed to differentiate iPSCs using protocol “iNeuron differentiation from human iPSCs.”

The authors describe procedure and equipment used for live-imaging of axonal cargoes. This was performed both using primary mouse cortical neurons and human iPSC-derived excitatory glutamatergic neurons. Equipment and software used varied based on laboratory site and scheduled upgrades to microscopy equipment during the course of this study.

The authors describe the procedure by which human iPSC-derived neurons or mouse embryonic fibroblasts (MEFs) were lysed and probed for levels of proteins of interest using Western blot.

This protocol describes RNP-based CRISPR/Cas9 gene-editing to generate knock-out iPSCs. iPSCs are transfected with sgRNA, recombinant Cas9, and GFP using Lipofectamine Stem. After FACS sorting to enrich for successfully transfected cells, individual clones are picked and expanded for further analysis.

The authors plate, culture, and transfect human iPSC-derived excitatory glutamatergic neurons for the purpose of observing transport of axonal cargoes under spinning disk confocal microscopy.

The authors fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal and axonal abundance of proteins of interest. For preceding culture of neurons, see “Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes.”