Generation of A549 PPM1H BromoTag CRISPR/CAS9 knock-in cell line
By Emma Sherrell onThis protocol details the generation of A549 PPM1H BromoTag CRISPR/CAS9 knock-in cell line.
Immunoblots
By Emma Sherrell onThe steps of this protocol are similar for different sources of protein such as cell lysate, cytosol, or membrane samples, and EV samples. However, some specific changes depend on the protein of interest, endogenous alpha-synuclein, or type of cells, hiPSC dopamine neurons.
shRNA knockdown
By Emma Sherrell onThis protocol describes a standard procedure to generate a stable cell line for a DNAJC5 knockdown using shRNA.
Differentiation of SH-SY5Y cells
By Emma Sherrell onThis protocol describes a standard procedure to differentiate SH-SY5Y cells into dopaminergic neurons using retinoic acid.
Mitochondria purification
By Emma Sherrell onThis protocol describes how to purify mitochondria from cell culture.
Co-immunoprecipitation
By Emma Sherrell onThis protocol describes a common procedure to perform co-immunoprecipitation.
Medium fractionation and EV preparation
By Emma Sherrell onThis protocol describes the characterization of the extracellular alpha-synuclein and DNAJC5.
CRISPR/Cas9 genome editing
By Emma Sherrell onThis protocol describes the generation of DNAJC5 KO using CRISPR/Cas9 edition.
In vitro depalmitoylation assay
By Emma Sherrell onThis protocol describes the in vitro depalmitoylation assay of DNAJC5.
Membrane and cytosol fractionation
By Emma Sherrell onThis protocol describes membrane and cytosol fractionation of cells expressing different DNAJC5 isoforms.
Strep-pull down assay
By Emma Sherrell onStrep pull down assay for FIP200(NTD)-TSF WT or mutants were co-transfected with GST-ATG13(363-517) and/or ULK1(MIT)-MBP.
Protein digestion and mass spectrometry analysis protocol
By Emma Sherrell onThis protocol details a protocol for the trypsin digestion of PI3KC3-C1 and subsequent analysis of digested peptides using LC-MS/MS.
Plasmid construction protocol
By Emma Sherrell onThis protocol details plasmid construction in general terms for the insertion of genes into the pCAG mammalian expression vector.
Preparing mitochondrial samples for immunoblot analysis
By Emma Sherrell onProtocol for preparation of mitochondrial samples for immunoblot analysis.
Molecular dynamics simulations
By Emma Sherrell onThis protocol details molecular dynamics simulations of PI3KC3-C1 on a lipid membrane.
HPLC analysis of nucleotides
By Emma Sherrell onIon-pair reversed phase HPLC analysis of the nucleotide bound to PI3KC3-C1.
Electrospray ionization analysis of eluted nucleotides
By Emma Sherrell onThis protocol describes the denaturation of PI3KC3-C1, and subsequent analysis of eluted nucleotides by electrospray ionization mass spec.
Cryo-EM sample preparation for PI3KC3-C1~RAB1A complex
By Emma Sherrell onThis protocol details cryo-EM sample preparation for PI3KC3-C1~RAB1A complex.
Cell culture
By Emma Sherrell onThis protocol describes routine cell culture maintenance as described in Adriaenssens & Nguyen et al., 2023 (BioRxiv).