Journal: Cell host & microbe

Article Title: ARIH2 is a Vif-dependent regulator of CUL5-mediated APOBEC3G degradation in HIV infection

doi: 10.1016/j.chom.2019.05.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-CUL5 (rabbit) Bethyl Cat# A302-173A Anti-CBFß (rabbit) Santa Cruz Biotechnology Cat# sc-56751 Anti-ELOB (rabbit) Abcam Cat# ab154854 Anti-ARIH2 (rabbit) Abcam Cat# EPR7670 Anti-ALIX (mouse) Biolegend Cat# 634502 Anti-HA (mouse) Covance Cat# 16B12 Anti-p24 (mouse) NIH AIDS Reagent Program Cat# 6521 Anti-vif (rabbit) NIH AIDS Reagent Program Cat# 809 Anti-HIV-1 Core Antigen Clone KC57 FITC (mouse) Beckmann Coulter Cat# 41116015 Anti-GAPDH (mouse) Sigma-Aldrich Cat# G8795-100UL Anti-Flag Hrp (mouse) Sigma-Aldrich Cat# A8592-1MG Anti-APOBEC3G (mouse) NIH AIDS Reagent Program Cat# 10069 Anti-Rabbit Hrp (goat) Bio-RAD Cat# 170-6515 Anti-Mouse Hrp (goat) Bio-RAD Cat# 172-1011 Anti-Goat Hrp (rabbit) Bio-RAD Cat# 172-1034 Anti-c-myc Sigma-Aldrich Cat# M4439 Anti-CD3 (UCHT1) Tonbo Biosciences Cat# 70-0038 Anti-CD28 (CD28.2) Tonbo Biosciences Cat# 70-0289 Bacterial and Virus Strains HIV-1 NL4-3 ΔEnv This study N/A HIV-1 NL4-3 ΔEnv ΔVif This study N/A HIV-1 NL4-3 Mulder et al., 2010 N/A HIV-1 NL4-3 ΔVif Stanley et al., 2012 N/A HIV-1 NL4-3 Nef-IRES Gfp Schindler et al., 2003 ; NIH AIDS Reagent Program Cat# 11349 Chemicals, Peptides, and Recombinant Proteins Anti-FLAG ® M2 Magnetic Beads Sigma Cat# M8823 3xFlag peptide ELIM Biopharm Custom order RapiGest SF Surfactant Waters Cat# 186001861 Sequencing grade trypsin Promega Cat# V5113 MG132 Calbiochem Cat# 474790 PolyJet transfection reagent SigmaGen Laboratories Cat# SL100688 Trans IT-293 Transfection Reagent Mirus Cat# MIR2700 PhosStop™ phosphatase inhibitors Roche Cat# 04906837001 cOmplete™ Protease Inhibitor Cocktail Roche Cat# 11836153001 Cas9 protein QB3 Macrolab, University of California, Berkeley Custom order Blasticidin Gibco Cat# R210-01 Zeocin Invitrogen Cat# R250-01 Doxycycline Clontech Cat# 631311 Human IL-2 IS Miltenyi Biotec Cat# 130-097-745 Puromycin Sigma-Aldrich Cat# P8833 1,10-Phenanthroline Sigma-Aldrich Cat# 131377 Polybrene (Hexadimethrine bromide) Sigma-Aldrich Cat# H9268 Polyethylenimine (PEI) Polysciences Cat# 23966-1 Deposited Data Shotgun proteomics RAW and analyzed data This study https://www.ebi.ac.uk/pride/archive/projects/PXD009012 Targeted proteomics RAW and analyzed data This study https://panoramaweb.org/project/UCSF%20-%20Krogan%20Lab/CRL5_HIV_interactome/begin.view?

Techniques: Virus, Recombinant, Magnetic Beads, Sequencing, Transfection, Protease Inhibitor, CRISPR, Software, Mass Spectrometry

Journal: Nature Communications

Article Title: Disrupting the α-synuclein-ESCRT interaction with a peptide inhibitor mitigates neurodegeneration in preclinical models of Parkinson’s disease

doi: 10.1038/s41467-023-37464-2

Figure Lengend Snippet: A Validation of PPI disruption using co-immunoprecipitation assays. Flag-CHMP2B was immunoprecipitated in the presence of HA-tagged A53T a-syn and GFP-tagged peptides. Negative controls were GFP alone (CTL) and a GFP-tagged scrambled version of PDpep1.3 (Scramble1.3); neither markedly co-immunoprecipitated with CHMP2B nor disrupted the interaction between CHMP2B and A53T a-syn. B Fluorescence polarization (FP) binding assay of FITC-labeled PDpep1.3 (blue) and displacement of the fluorescent peptide with increasing concentrations of a-syn (black). Error bars represent ± s.d. of the fit ( n = 3) (left panel). FP binding assay of FITC-labeled a-syn peptides. Error bars represent ± s.d. of the fit ( n = 3) (right panel). C PDpep1.3 restores reduced LAMP1 expression due to A53T a-syn in HEK293 cells as shown by confocal micrographs (scale bars = 15 μm). Representative immunoblots of D LAMP1 levels or E CD63 levels in HEK293 cells upon co-transfection of A53T a-syn and peptides. Controls were GFP alone (pLJM1) and a GFP-tagged scrambled version of the initial peptide (scramble). Loading control was beta-actin. Experiments were done in triplicate. F Endolysosomal flux assay using flow cytometry in HEK293 cells co-transfected with A53T a-syn or control vector plus Scramble1.3 or PDpep1.3. Cells were treated with the lysosomal inhibitor Leupeptin (Leu) as indicated. Data represent means ± s.d. (unpaired two-tailed t -test, t (4) = 2.776; A53T+PDpep1.3, P = 0.0010; n = 3). G Autophagy flux assay in HEK293 cells stably expressing a luminescent LC3-HiBiT reporter co-transfected with A53T a-syn or empty vector (EV) plus Flag-Scramble1.3 or Flag-PDpep1.3. Cells were treated with the lysosomal inhibitor Bafilomycin A1 or the autophagy inducer Rapamycin as indicated. Bars represent means ± s.e.m. ( n = 3). H Representative images of cortical neurons transduced with A53T a-syn or EV plus Scramble1.3-RFP or PDpep1.3-RFP (scale bars = 5 μm). I Quantification of LAMP1 fluorescence in RFP-positive neurons. Bars represent means ± s.e.m. (nested one-way ANOVA, F (3, 102) = 4.430; P = 0.0057; n = 3). J The proposed mechanism of PDpep1.3 is that the peptide disrupts the a-syn-CHMP2B interaction to restore degradation of a-syn by the endolysosomal pathway (created using Servier Medical Art at https://smart.servier.com/ ). * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Lentiviruses were made in a 15 cm dish format by transfecting packaging cells (HEK293T) with a three-plasmid system (expression vector, psPAX2 and pMD2.G) and using Polyjet DNA transfection reagent (FroggaBio, SL100688.1).

Techniques: Immunoprecipitation, Fluorescence, FP-binding Assay, Labeling, Expressing, Western Blot, Cotransfection, Flux Assay, Flow Cytometry, Transfection, Plasmid Preparation, Two Tailed Test, Stable Transfection, Transduction