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: JCI Insight

Article Title: Repression of AXL expression by AP-1/JNK blockage overcomes resistance to PI3Ka therapy

doi: 10.1172/jci.insight.125341

Figure Lengend Snippet: (A) RNA sequencing data obtained from isogenic BYL719 sensitive vs. acquired resistance CAL33 and KYSE180 cells (HNSCC and ESCC, respectively). Presented in the Venn diagram are TF signatures upregulated in BYL719-resistant cells and their overlap with binding sites for TF in the promoter of AXL. (B) WB analysis of CAL33 and KYSE180 BYL719-sensitive vs. -resistant cells showing the expression of TFs (l.e., long exposure; s.e., short exposure). (C) A qPCR analysis comparing the mRNA levels of AXL and c-JUN in CAL33- and KYSE180-sensitive vs.-resistant cells (n > 10). (D) IF images of CAL33- and KYSE180 BYL719–sensitive vs. –resistant cells showing c-JUN (CY-3-labeled) in red, AXL (Alexa488-labeled) in green, and DAPI in blue. Scale bar: 100 μm. (E) Upper panel: WB analysis showing AXL levels after transfection with siRNA for the silencing of c-JUN and c-FOS. Lower panel: qPCR analysis showing AXL mRNA levels in cells transfected with siRNAs for the silencing of c-JUN and c-FOS (n > 6). All WB analysis was assessed in 2–4 independent experiments. All qPCR analysis was assessed in 2–4 independent experiments. One-way ANOVA P value is shown. **P < 0.01.

Article Snippet: For the production of shRNAs cell lines, we created lentiviruses by transfecting HEK293 cells with the viral plasmids psPAX2, pMD2.G, and PLKO with shRNAs — a control scrambled sequence (shCT) or 2 different sequences for the silencing of AXL expression (shAXL1 and shAXL2, MSKCC RNAi core facility identifiers TRCN0000001037 and TRCN0000194771, respectively) using PolyJet transfection reagent (SignaGen, SL100688) according to the manufacturer’s protocol.

Techniques: RNA Sequencing Assay, Binding Assay, Expressing, Labeling, Transfection

Journal: JCI Insight

Article Title: Repression of AXL expression by AP-1/JNK blockage overcomes resistance to PI3Ka therapy

doi: 10.1172/jci.insight.125341

Figure Lengend Snippet: (A) Viability was assessed in cell lines treated with escalating doses of BYL719 for 4 days. Analysis of BYL719 IC50 values in HNSCC and ESCC cells following transfection with siRNAs to silence c-JUN and c-FOS expression (n > 6). (B) Viability was assessed in cell lines treated with BYL719 and SP600125 for 4 days. Analysis of BYL719 IC50 values following JNK inhibition with SP600125 (5 and 10 μM) in HNSCC and ESCC cells (n > 6). (C) WB analysis showing AXL level and AKT/mTOR pathway activation in HNSCC and ESCC cells treated with BYL719 (2 μM), SP600125 (5 and 10 μM), and the combination therapy for 24 hours. (D) qPCR analysis of AXL mRNA levels in cells treated as in with BYL719 (2 μM), SP600125 (10 μM), and combination for 24 hours (n > 6). (E) Viability was assessed in cell lines treated with escalating doses of BYL719 and SP600125 for 4 days. Synergy test for the interaction between BYL719 and SP600125. The synergy test was analyzed using Chalice software (Horizon), and a synergy score was extracted. All WB analysis was assessed in 2–3 independent experiments. All viability experiments were assessed in 2–3 independent experiments. All qPCR experiments were assessed in 2–3 independent experiments. One-way ANOVA P values are shown. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: For the production of shRNAs cell lines, we created lentiviruses by transfecting HEK293 cells with the viral plasmids psPAX2, pMD2.G, and PLKO with shRNAs — a control scrambled sequence (shCT) or 2 different sequences for the silencing of AXL expression (shAXL1 and shAXL2, MSKCC RNAi core facility identifiers TRCN0000001037 and TRCN0000194771, respectively) using PolyJet transfection reagent (SignaGen, SL100688) according to the manufacturer’s protocol.

Techniques: Transfection, Expressing, Inhibition, Activation Assay, Software

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

Journal: Cell reports

Article Title: A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity

doi: 10.1016/j.celrep.2019.01.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: In brief, cells were transfected with 1 μg pLentiCMV DEST plasmid + 0.25 mg of each of three 3 rd generation lentiviral packaging plasmids (pMDLg/pRRE, pRSV-Rev and pMD2.G, Addgene plasmids #12251, #12253 and #12259, respectively, from Didier Trono) using PolyJet DNA Transfection Reagent (SignaGen Laboratories, Cat# SL100688 Rockville, MD) as per the manufacturer’s instructions.

Techniques: Virus, Recombinant, In Vitro, Transfection, DNA Extraction, RNA Extraction, Reverse Transcription, Purification, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Bradford Assay, Control, Software

Journal: iScience

Article Title: Designer polyQ fusion proteins sequester USP7/HDM2 for modulating P53 functionality

doi: 10.1016/j.isci.2025.112025

Figure Lengend Snippet:

Article Snippet: PolyJet reagent , SignaGen , Cat# SL100688.

Techniques: Virus, Recombinant, Protease Inhibitor, Reverse Transcription, SYBR Green Assay, Sequencing, Software