Plasmid: pLV[Exp]-EF1A>V5/hKAT8[NM_032188.3]:IRES:Puro
By onPlasmid: Human KAT8 gene with N-terminal V5 tag cloned into the pLV plasmid backbone under a EF1a promoter and including IRES puromycin selection cassette by Vectorbuilder.
Cell line construction and maintenance for Lyso-IP and Endo-IP analysis of amyloid precursor protein processing
By onLyso-IP is a method that allows for the isolation of lysosomes for proteomics and metabolomics (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.bx9hpr36). We have developed an analogous approach for purification of early/sorting endosomes (Endo-IP). In addition, we have found that endolysosomal purification via Lyso-IP and Endo-IP can be coupled with a quantitative proteomics workflow to obtain snapshots of Amyloid Precursor Protein (APP) processing to its Aβ products (Park et al. in submission). Here, we describe methods for cell line construction and maintenance of 293 cells with TMEM192-3xHA and 3xFLAG-EEA1, which are used for lysosome and endosome purification, respectively, with the addition of patient mutations to APP promotes processing. Cells with endogenously tagged TMEM192 and stably expressing FLAG-EEA1 are referred to as 293EL cells, for Endo-IP and Lyso-IP. These cells were also prepared in a form that has a deletion of the APP gene (293EL;APP-/-) and the same cells reconstituted with a lentivirus stably expressing APPSw;T700N to allow functional analysis of APP processing.
pCAG-OSF-ATG13 (2-197)-W50D
By onPlasmid for expression of human ATG13 HORMA W50D mutant in mammalian cells.
Adhesive Removal Test to assess sensorimotor deficits in parkinsonian mice
By onThis behavior is used to assess fine motor movements in a mouse Parkinsonian model. It checks for correct paw and mouth sensitivity (time-to-contact) and correct dexterity (time-to-remove).
Structure and activation of the human autophagy-initiating ULK1C:PI3KC3-C1 supercomplex
By onAuthors determine cryo-EM structure of human ULK1C core and its complex with PI3KC3-C1. ULK1C core undergoes a rearrangement from 2:1:1 to 2:2:2 stoichiometry, suggesting a structural mechanism for autophagy initiation.
GFP-TBK1: expression and purification
By onThis protocol describes how to express and purify human TBK1 tagged N-terminally with eGFP.
The Hsc70 disaggregation machinery removes monomer units directly from α-synuclein fibril ends
By onUsing microfluidic diffusional sizing, the authors show that the molecular chaperone family Hsp70 (specifically Hsc70, DnaJB, and Apg2) can completely dissolve alpha-synuclein aggregation and revert it back to its monomeric state.
Post mortem human substantial nigra TH staining
By onThis protocol is standardized for postmortem (frozen) SN tissue from pathologically diagnosed PD patients and control individuals for Immunofluorescence staining.
A549 PPM1H-BromoTag (CVCL_C8YV)
By onCell Line: PPM1H-BromoTag CRISPR/CAS9 A549 knock-in cell line generated from parental A549 cells (ATCC A549 CCl-185) by introducing a BromoTag linked to the C-terminus of PPM1H using CRISPR/Cas9 gene-editing technology.
Phenotypic effect of GBA1 variants in individuals with and without Parkinson disease: the RAPSODI study
By onThe authors’ results support previous evidence that GBA1-positive PD has a specific phenotype with more severe non-motor symptoms. The authors did not reproduce previous findings of more frequent prodromal PD signs in non-affected GBA1 carriers.
ImageJ FIJI code to semi-automatize the morphological analysis of microglia in histological sections
By onImageJ FIJI code to semi-automatize the morphological analysis of microglia in histological sections.
Immunohistochemistry of AAV-treated tissues
By onOutlines procedures to perform immunohistochemistry on tissue that had been treated with AAV.
Passaging of hPSCs grown on MEFs
By onThis protocol describes the standard procedure of using collagenase to passage human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).
Unilateral intranigral AAV alpha synuclein mouse model
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Intracellular Cytokine (ICS) Staining Protocol
By onThis protocol details about intracellular cytokine (ICS) staining.
H9 ES AAVS1-NGN2 FAM134C/A/B-/-; PiggyBac-Keima-REEP5
By onES cells were modified to create iNeurons lacking FAM134C, A, B receptors and expressing Keima-REEP5 ER-phagy flux reporter. CRISPR/Cas9 was used to introduce NEUROG2 construct in AAVS1 locus and knockout RETREG1, 2, 3 genes.
