Journal: Cell reports

Article Title: Pyruvate metabolism controls chromatin remodeling during CD4 + T cell activation.

doi: 10.1016/j.celrep.2023.112583

Figure Lengend Snippet: Figure 2. ACSS2 inhibition or ACLY deficiency does not affect epigenome remodeling during T cell activation (A) Immunoblots of ACLY, ACSS2, PDCE1, and actin from human T cells activated for 8, 16, 24, or 48 h with anti-CD3/CD28 stimulation. (B and C) CD25 protein expression was measured by flow cytometry (FACS) and IL2ra gene expression was determined by quantitative reverse transcription- polymerase chain reaction (qRT-PCR) in human CD4+ T cells activated with anti-CD3/CD28 in the presence and absence of ACSS2 inhibitor (15.6 mM).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER FCCP, mitochondrial oxidative phosphorylation uncoupler Abcam # ab120081 Rotenone Merck Millipore # R8875 UK-5099 (Synonyms: PF-1005023) Med Chem Express # HY-15475 3PO R98% (HPLC) Sigma-Aldrich # SML1343 Sodium acetate Sigma-Aldrich # S1429 DCA Tocris Bioscience # 2755 Etomoxir sodium salt hydrate Sigma-Aldrich # E1905 ACSS2 inhibitor Selleck # S8588 6,8-Bis(benzylthio)-octanoic acid Sigma-Aldrich # SML0404 Sodium Pyruvate Thermo Fisher # 11360070 SB204990 Med Chem Express # HY-16450 BMS303141 Sigma-Aldrich # SML0784 HEPES Sigma-Aldrich #H4034-100G EDTA Sigma-Aldrich #EDS-100G DL-Dithiothreitol solution (DTT) Sigma-Aldrich #43816-10ML Bovine serum albumin (BSA) Sigma-Aldrich #A9647-500G D-(+)-Glucose solution Sigma-Aldrich #G8769-100ML 13C6-glucose Cambridge Isotope Lab #CLM-1396 DMSO Sigma-Aldrich # D2650 Ficoll Paque Plus Sigma-Aldrich # GE17-1440-02 13C6-glucose Cambridge Isotope Lab #CLM-1396 Cell Trace Violet Invitrogen #C34557 Molecular ProbesTM 2-NBDG Invitrogen #N13195 SYBR Thermo fisher #S33102 iScript cDNA kit biorad #170-8891 Formaldehyde Sigma-Aldrich #252549-1L DAPI (1:2000) Invitrogen #D1306 Zombie NIRTM Fixable Viability Kit Biolegend #423106 Zombie GreenTM Fixable Viability Kit Biolegend #423112 7-AAD Invitrogen #A1310 D-(+)-Glucose solution Sigma-Aldrich #G8769-100ML GolgiPlugTM (Protein Transport Inhibitor) BD Biosciences #555029 Tween 20 Sigma-Aldrich #P7949-500ML HALT protease inhibitor Thermo scientific #78439 Powdered milk Carl Roth #T145.2 PierceTM Protein A/G Magnetic Beads thermo fisher #88802 Proteinase K, recombinant, PCR Grade Sigma #3115828001 RNeasy Mini Kit Qiagen #74106 Seahorse XF DMEM medium Agilent Technologies #103575-100 Critical commercial assays MagniSortTM Human CD4+ T cell Enrichment Kit eBioscience #8804-6811-74 CD4 (L3T4) MicroBeads, mouse Miltenyi # 130-117-043 Duolink In Situ Detection Reagents Red Sigma DUO92008 Duolink Green PLA Sigma # DUO92014 Foxp3/Transcription Factor Staining Buffer Set eBioscience #00-5523-00 BD Cytofix/Cytoperm Fixation/Permeabilization kit BD Biosciences #554714 seahorse XFe24 Flux Packs Agilent #1023-40-100 Acetyl-CoA Assay Kit Biovision, Milpitas CA #K317-100 (Continued on next page) Cell Reports 42, 112583, June 27, 2023 19

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Cytometry, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Preparation and characterization of the cell lines used in the present study. (A) Detection of SMS1, SMS2, and GAPDH in JAR, JAR4, JEG3, and HeLa-mCAT#8 cell lines by immunoblotting. (B) Nucleotide sequences around the target region for the SGMS1 gene-specific sgRNA in exon 9 of the SGMS1 gene of the SMS1KO22 clone (clone 22) were aligned with those of the parental JAR4 cells (parent). Target sequences for the sgRNA and following protospacer adjacent motifs are indicated by underlined and boxed sequences of the parental JAR4 cells, respectively. The SMS1KO22 clone has a homozygous 7-nucleotide deletion in the SGMS1 gene. (C) Detection of SMS1, SMS2, and GAPDH in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2 by immunoblotting. (D) Detection of clustered SM on the surface of JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Cells were treated with EGFP-NT-lysenin and analyzed by flow cytometry. Histograms with magenta line and gray fill represent EGFP-NT-lysenin-treated and untreated cells, respectively. Cells in the M1 region (fluorescent intensity of 50 and above) were defined as positive for binding to EGFP-NT-lysenin. The means and standard deviations of triplicate samples are reported in panel E. (E) Significant differences as determined by one-way analysis of variance (ANOVA) with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant. (F) Quantification of sphingolipids in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2 by LC-MS analysis. Lipids were extracted from cells and quantified by LC-MS. The graphs indicate the means and standard deviations of triplicate samples. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. CMH, ceramide monohexoside (glucosylceramide and galactosylceramide); CDH, ceramide dihexoside (lactosylceramide and galabiosylceramide); Gb3, trisaccharide globo-series sphingolipid; GM3, monosialodihexosylganglioside.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Western Blot, Derivative Assay, Flow Cytometry, Binding Assay, Comparison, Liquid Chromatography with Mass Spectroscopy

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Impacts of SGMS1 gene knockout on RuV growth. (A) Growth kinetics of RuV in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Supernatants of each cell line inoculated with the RuV TO-336WT strain at an MOI of 10 were harvested at 0, 1, 2, 3, or 4 days after inoculation. Infectious titers in the supernatants are represented as means and standard deviations of triplicate samples. (B) Fluorescent microscopy images of each cell line, NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, or SMS1KO22/SMS2, inoculated with the RuV TO-336WT strain at 3 days after inoculation. Green signals indicate the expression of the p150-AG1 protein. Nuclei were stained by DAPI (blue). (C) The rate of p150-AG1-expressing cells inoculated with the RuV TO-336WT strain under the same conditions as for panel B. Cells detached with trypsin-EDTA and fixed with 4% paraformaldehyde were analyzed by flow cytometry. The graph indicates the means and standard deviations of triplicate samples. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: *, P < 0.05; ****, P < 0.0001; ns, not significant. (D and E) Growth kinetics of SINV (D) and MeV (E) in NT1 or SMS1KO22 cells. Infectious titers of progeny viruses are represented as means and standard deviations of triplicate samples.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Gene Knockout, Derivative Assay, Microscopy, Expressing, Staining, Flow Cytometry, Comparison

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Impacts of knockout of the SGMS1 or SGMS2 gene on infectivity and entry of RuV in HeLa cells. (A) Detection of SMS1, SMS2, and GAPDH in HeLa-mCAT#8 cell line (Parent) and its gene-edited clones, SGMS1 or SGMS2 single-knockout (ΔSMS1 or ΔSMS2) and double-knockout (DKO) cells, by immunoblotting. (B) Growth kinetics of RuV in HeLa-derived cell lines. Each cell line was inoculated with the TO-336WT-AG1 RuV strain at an MOI of 10, and the supernatants were harvested at the indicated days after incubation. The infectious titers of RuV in the supernatants are represented as means and standard deviations of triplicate samples. (C) Infectivity of pseudotyped VSV in each cell line. Each cell line was inoculated with firefly luciferase gene-coding pseudotyped VSVs VSVFLuc-ΔG (ΔG), VSVFLuc-RV/CE2E1 (RuV-CE2E1), or VSVFLuc-G (VSV-G). The firefly luciferase activity was measured at 24 h postinoculation. The graph indicates the means and standard deviations of three independent assays. Significant differences as determined by two-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Knock-Out, Infection, Clone Assay, Double Knockout, Western Blot, Derivative Assay, Incubation, Luciferase, Activity Assay, Comparison

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Impacts of SGMS1 gene knockout on genome replication, entry, and binding of RuV. (A) Reporter assay of the RuV-subgenomic replicon. NT1 or SMS1KO22 cell line was transfected with the subgenomic replicon RNA HS-Rep-P2R (SGR) or replication-defective mutant HS-Rep-GND-P2R (GND), which expressed Rluc as a reporter, together with mRNAs encoding the RuV-C protein and firefly luciferase (Fluc). After 72 h of transfection, RLuc activity was determined and normalized by Fluc activity. The graph indicates the means and standard deviations of three independent assays. Significant differences as determined by two-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant. (B) Infectivity of pseudotyped VSVs in JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Each cell line was inoculated with Fluc gene-encoding pseudotyped VSVs, VSVFLuc-ΔG (ΔG), VSVFLuc-RV/CE2E1 (RuV-CE2E1), or VSVFLuc-G (VSV-G). The Fluc activity was measured at 24 h postinoculation. The graph indicates means and standard deviations of three independent assays. Significant differences by two-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: ****, P < 0.0001; ns, not significant. (C) Binding of RuV to JAR4-derived cell lines NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, and SMS1KO22/SMS2. Each cell line was incubated with RuV at an MOI of 4 on ice for 1 h and then washed to remove unbound viruses. Total RNA was extracted from the cells, and the amount of RuV genomic RNA was determined by quantitative RT-PCR and normalized by the amount of total RNA. The graph indicates the means and standard deviations of three independent assays. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: **, P < 0.01; ****, P < 0.0001; ns, not significant.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Gene Knockout, Binding Assay, Reporter Assay, Transfection, Mutagenesis, Luciferase, Activity Assay, Comparison, Infection, Derivative Assay, Incubation, Quantitative RT-PCR

Journal: mBio

Article Title: Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry

doi: 10.1128/mbio.01698-22

Figure Lengend Snippet: Penetration of the RuV genome into the cytoplasm. NT1 or SMS1KO22 cells (A and B), or SMS1KO22cells, SMS1KO22/SMS1-WT clone 1, or SMS1KO22/SMS2 clone 1 (C and D) were inoculated with RuV. After incubation at 37°C for 3 h, cells were fixed with 4% paraformaldehyde. For the control experiment in panel A, NT1 cells were incubated with a medium containing BAPTA-AM (final concentration, 50 μM) before inoculation of RuV at 1 h. The RuV genome (pseudocolored in magenta) and the E1 protein (green) were stained by in situ hybridization and indirect immunofluorescence assay, respectively. Nuclei were stained by DAPI (blue). In panels A and C, representative z -stack images are shown. The three columns on the right are enlarged images of the areas enclosed by dashed boxes in the left column. Bars for original and enlarged images indicate 20 μm and 5 μm, respectively. In panels B and D, percentages of puncta in which RuV genomic RNA is present and colocalized with E1 protein are indicated. The graphs indicate the means and standard deviations of three independent assays. Significant differences by one-way ANOVA with Tukey’s post hoc multiple-comparison tests are indicated by asterisks: **, P < 0.01; *, P < 0.05.

Article Snippet: Anti-SMS1 rabbit polyclonal antibody (HPA045191; Atlas Antibodies, Bromma, Sweden), anti-SMS2 mouse monoclonal antibody (7D10; Santa Cruz Biotechnology, Dallas, TX), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (3H12; MBL, Nagoya, Japan), anti-RuV E1 protein mouse monoclonal antibody (2Q2070; US Biologicals, Salem, MA), anti-VSV-G protein mouse monoclonal antibody (8G5F11; Kerafast, Boston, MA), anti-MAVS rabbit monoclonal antibody (D5A9E; Cell Signaling Technology, Danvers, MA), anti-PKR rabbit monoclonal antibody (D7F7; Cell Signaling Technology), anti-p230 trans-Golgi (also known as GOLGA4) mouse monoclonal antibody (clone 15; BD Biosciences, Franklin Lakes, NJ), anti-4F2hc/CD98 (also known as SLC3A2) rabbit monoclonal antibody (Cell Signaling Technology), and anti-dsRNA mouse monoclonal antibody (J2; English and Scientific Consulting Kft., Szirak, Hungary) were used in this study.

Techniques: Incubation, Control, Concentration Assay, Staining, In Situ Hybridization, Immunofluorescence, Comparison

Journal: Cell Death & Disease

Article Title: Opposing roles for GSK3β and ERK1-dependent phosphorylation of huntingtin during neuronal dysfunction and cell death in Huntington’s disease

doi: 10.1038/s41419-025-07524-0

Figure Lengend Snippet: A Representative images from healthy/normal (WT, Q17) or diseased (HD, Q109) human iPSCs stained with the pluripotent marker OCT-4, the neuronal precursor (NPC) marker Nestin and the mature neuronal markers MAP2 and βIII-Tubulin. Hoechst stains nuclei. Scale = 25 μm. Differentiated neurons show Synaptophysin (SYP) positive staining. Scale = 10 μm. B Electrophysiological analysis of WT and HD human neurons differentiated from iPSCs show action potentials, which are abolished in the presence of TTX or TEA. C Schematic diagram of human iNeuron lysate fractionation into perinuclear supernatant (PNS), light membrane (LM), soluble (SF), and heavy membrane (P1) fractions by ultra-centrifugation and sucrose gradient separation. D Workflow for quality control and quantification of unique peptides identified from LC-MS of HTT-IPs from WT or HD human iNeurons. E Hierarchical cluster heat map showing the avg. relative abundance (spectral count; SpC) of 800 proteins (≥3 unique peptides/trial across ≥2 biological replicates) quantified across the WT and HD HTT-IPs with a normalized fold change (FC) threshold of ±2X and a significance threshold of p < 0.05 determined by a Welch’s t test across three independent biological replicates. Increased in HD HTT-IP = red, decreased in HD HTT-IP = blue. In addition, proteins were identified in only WT HTT-IP (lost = green) or in only HD HTT-IP (gained = orange). F Volcano plot with the y axis depicting significance (−log 10 [ p value]) and the x axis depicting fold change of individual peptides between HD and WT HTT-IPs (log 2 [FC]). Three independent biological replicates were performed for each genotype. A negative, no-antibody IP was performed to account for non-specific peptide association with magnetic beads. G Representative western blot of HTT-IP from WT or HD LMs, probed against HTT, KIF5A, KIF5B, KIF5C, DNCT, MAP1B, MAP2, RAB2, RAB5, RAB7, VPS35, or SUMO2. Except for KIF5A, all show presence in WT and HD HTT-IP. No bands are seen in the negative no antibody control (−Crtl). n = 3. Statistical analysis was conducted using the two-sample two-sided Student’s t test comparing signal/noise intensity between bands in WT and HD conditions normalized to WT. Data represented as mean ± SEM. ns = p > 0.05, * p < 0.05, ** p < 0.005.

Article Snippet: Blots were blocked using TBST with 5% BSA for 60 mins at 25 °C and incubated with primary antibodies (SYT1 (Thermofisher 1:1000), Rab4 (Abcam 1:1000), Rab5 (Abcam 1:1000), Rab2 (SCBT 1:500), Rab7 (SCBT 1:500), VPS35 (SCBT 1:500), SUMO2/3 (Cytoskeleton 1:500), KIF5A (Goldstein 1:250), KIF5B (Goldstein 1:250), KIF5C (Goldstein 1:250), DIC (Abcam 1:1000), DNCT (Abcam 1:1000), Actin (ThermoFisher 1:1000), Tubulin (Abcam 1:2000), HTT rabbit polyclonal (Abcam 1:1000), HTT mouse monoclonal (EMD Millipore 1:1000), Golgi (Millipore Sigma 1:1000), Cytochrome C (Santa Cruz 1:1000), TOM20 (CellSignaling Technology 1:500), MAP1B (SCBT 1:1000), MAP2 (BD Pharmigen 1:1000), Total AKT1 (CellSignaling Technology 1:1000), pAKT1 (Ser473, CellSignaling Technology 1:1000), Total GSK3α/β (CellSignaling Technology 1:1000), pGSK3α/β (pY279/pY216; Abcam 1:1000), or ERK (pan-ERK; BD Transduction Laboratories 1:1000) for 16 h at 4 °C.

Techniques: Staining, Marker, Fractionation, Membrane, Centrifugation, Control, Liquid Chromatography with Mass Spectroscopy, Magnetic Beads, Western Blot

Journal: Cell Death & Disease

Article Title: Opposing roles for GSK3β and ERK1-dependent phosphorylation of huntingtin during neuronal dysfunction and cell death in Huntington’s disease

doi: 10.1038/s41419-025-07524-0

Figure Lengend Snippet:

Article Snippet: Blots were blocked using TBST with 5% BSA for 60 mins at 25 °C and incubated with primary antibodies (SYT1 (Thermofisher 1:1000), Rab4 (Abcam 1:1000), Rab5 (Abcam 1:1000), Rab2 (SCBT 1:500), Rab7 (SCBT 1:500), VPS35 (SCBT 1:500), SUMO2/3 (Cytoskeleton 1:500), KIF5A (Goldstein 1:250), KIF5B (Goldstein 1:250), KIF5C (Goldstein 1:250), DIC (Abcam 1:1000), DNCT (Abcam 1:1000), Actin (ThermoFisher 1:1000), Tubulin (Abcam 1:2000), HTT rabbit polyclonal (Abcam 1:1000), HTT mouse monoclonal (EMD Millipore 1:1000), Golgi (Millipore Sigma 1:1000), Cytochrome C (Santa Cruz 1:1000), TOM20 (CellSignaling Technology 1:500), MAP1B (SCBT 1:1000), MAP2 (BD Pharmigen 1:1000), Total AKT1 (CellSignaling Technology 1:1000), pAKT1 (Ser473, CellSignaling Technology 1:1000), Total GSK3α/β (CellSignaling Technology 1:1000), pGSK3α/β (pY279/pY216; Abcam 1:1000), or ERK (pan-ERK; BD Transduction Laboratories 1:1000) for 16 h at 4 °C.

Techniques: Transduction, Recombinant, Protease Inhibitor, Magnetic Beads, Plasmid Preparation, In Situ, Software, Imaging

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) A schematic depicting our strategy to generate GBA1 variants with enhanced secretion. Endogenous signal sequence of human GBA1 was swapped with signal sequences from highly secreted proteins. Top 4 signal sequences (SS) were narrowed down using in-silico tools that predicted robust secretion as well as high (>96%) probability of cleavage at the end of signal sequence. (B) HEK293T cells were transfected with GBA1 plasmid containing the indicated SS variants, lysed, and GCase enzyme activity was determined in cell lysates. Mean ± SEM, n = 4 independent experiments. Untransfected versus all GBA1 constructs as well as GFP transfected versus all GBA1 constructs: ***p<0.001; One-way ANOVA with Turkey’s multiple comparison test. (C) Representative immunofluorescence images demonstrating co-localization of active GCase and lysosomes. HEK293T cells were transfected with GBA1 variant constructs, incubated with MDW933 fluorescence probe to label active GCase, and immunostained with Lamp1 antibody for lysosomes. Blue and yellow arrowheads point to individual puncta showing co-localization of GCase with Lamp1. Scale bar is 10 microns. (D and E) HEK293T cells were transfected with GBA1 variant constructs and cell culture media was collected to detect secreted GCase (D) and measure GCase enzyme activity (E). Mean ± SEM, n = 4 independent experiments. Untransfected versus all GBA1 constructs as well as GFP transfected versus all GBA1 constructs: ***p<0.001; One way-ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Sequencing, In Silico, Transfection, Plasmid Preparation, Activity Assay, Construct, Comparison, Immunofluorescence, Variant Assay, Incubation, Fluorescence, Cell Culture

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: AAV GMU01 capsid expressing the indicated GBA1 variants were administered by bilateral ICV to 4 month-old C57/BL6 mice at 1e11 vector genomes per animal and 5 μl per hemisphere. N=4 animals per group. The sagittal sections of brain hemisphere were analyzed 4 weeks post-injection. (B) Representative images demonstrating GCase secretion with engineered variants. Vector biodistribution is shown with in situ hybridization to WPRE (top panels) and GCase is shown with immunohistochemistry. (C) Representative images demonstrating cross-correction. Higher magnification images of WPRE mRNA (left) and GCase protein (right) in SS3-GBA1 injected mice brain, image corresponds to the purple box in (A). Cells positive for both WPRE mRNA and GCase are shown in red arrows (AAV-transduced cells) while mRNA negative and GCase positive cells are shown in green arrows (cross-corrected cells). Scale bar is 500 microns.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Expressing, Plasmid Preparation, Injection, In Situ Hybridization, Immunohistochemistry

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Measurement of Lyso-GL1 lipid substrate levels by LC-MS in cortex and liver of C57/BL6 mice 24 hours post CBE injection by IP injection at indicated doses. N=7 animals for CBE 0 mg/kg and N=4 per all the other groups. Data are Mean ± SEM. (B) Kinetic analysis of Lyso-GL1 lipid substrate by LC-MS in cortex and liver of 8-month-old mice from three different genotypes ( Gba D409V/D409V , Gba D409V/+, and Gba +/+ ). Mice with 100 mg/kg CBE IP dosing. Data are Mean ± SEM. 8 – 10 animals at each time point. (C) Study design: AAV GMU01 capsid expressing the indicated GBA1 variants were administered by bilateral ICV to 4-month-old C57/BL6 mice at 1e11 vector genomes per animal and 5μl per hemisphere. N=8 animals per group. CBE (conduritol β-epoxide) was administered via IP injection at 100 mg/kg 24 hours prior to necropsy. (D) Representative image of in situ hybridization to WPRE mRNA from the analyzed sagittal sections. Cortex is proximal to site of injection and cerebellum is distal to site of injection. (E-G) Measurement of Lyso-GL1 lipid substrate levels by LC-MS in midbrain (E), cerebellum (F), and hindbrain (G). No CBE group in the graphs shown is control mice that did not receive any AAV vector or CBE injection. 8 mice per group. ***p<0.001, **p<0.01, *p<0.05; One way- ANOVA with Tukey’s multiple comparisons test with all groups compared to CBE-treated vehicle injected group.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Liquid Chromatography with Mass Spectroscopy, Injection, Expressing, Plasmid Preparation, In Situ Hybridization, Control

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: 2-3 years old cynomolgus monkeys (2-3kg) were dosed either with AAV GMU01-WT-GBA1 or AAV GMU01-SS3-GBA1 at 1.25e13 vector genomes per animal by direct injection to the cisterna magna (ICM) in 2.5 ml volume at 0.125 ml/min rate. 6 weeks post- dosing, 30 mg/kg of CBE was administered by IV injection 48 hours prior to necropsy. Samples represent 64 brain biopsy punches encompassing 19 distinct grey matter regions and 7 distinct white matter regions. (B and C) AAV vector genome copies determined from 64 brain biopsy punches by Gba1 dPCR and normalized to the Tubb3 gene copy number to obtain VG copies per cell (B). Transgene (mRNA) expression measured by GBA1 RT-dPCR normalized to endogenous Hprt gene (C). Median with inter-quartile range across 64 punches representing 19 grey matter and 7 white matter regions. (D) Correlation of vector genome with transgene expression (mRNA) was determined between WT-GBA1 and SS3-GBA1. Each data point is average of all NHPs for that punch. Non- parametric Spearman’s rank correlation. (E) Lyso-GL1 changes in plasma pre-AAV, post-AAV and pre-CBE, and at necropsy across all NHPs. (F and G) Lyso-GL1 level (F) and C18 GL1 level (G) across 64 brain biopsy punches. Each data point is average of all NHPs in the group for that punch. ***p<0.001; Two way-ANOVA with Tukey’s multiple comparisons test. (H and I) Multiplex fluorescent imaging assay with in situ hybridization for mRNA and immunohistochemistry for GBA1 protein and cell marker. Low magnification image showing stained motor cortex region of SS3-GBA1 injected NHP and zoomed-in images corresponding to the white box (H). Triplex to determine identity of cross-corrected cells using specific cell type markers, NeuN for neurons, S100b for astrocytes, and Iba1 for microglia (I). Yellow arrowheads point to AAV-transduced cells and red arrowheads point to cross-corrected cells. Scale bar is 20 microns.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Plasmid Preparation, Injection, IV Injection, Expressing, Clinical Proteomics, Multiplex Assay, Imaging, In Situ Hybridization, Immunohistochemistry, Marker, Staining

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: 2-3 years old cynomolgus monkeys (2-3kg) were administered with 3 different doses of AAV GMU01-SS3-GBA1 by direct injection to the cisterna magna (iCM) in 2.5 ml volume at 0.125 ml/min rate, 2.5e12 vector genomes per animal (low dose), 7.5e12 vector genomes per animal (mid dose), and 2.5e13 vector genomes per animal (high dose). 8 weeks post-dosing, 30 mg/kg of CBE was administered by IV injection 24 hours prior to necropsy. Samples represent 64 brain biopsy punches encompassing 19 distinct grey matter regions and 7 distinct white matter regions. (B and C) Assessment of vector genomes in dose-range finding study with 3 doses tested, 5 NHPs per group. Median with inter-quartile range across 64 punches representing 19 grey matter and 7 white matter regions (B). Same data shown across different brain regions (C). T/I/C: temporal/insulate/cingulate (D) GCase enzyme activity in dose-range finding study with 3 doses tested, N=5 NHPs per group. Median with inter-quartile range across 64 punches representing 19 grey matter and 7 white matter regions. **p<0.05, ***p<0.001; Two way-ANOVA with Tukey’s multiple comparisons test. (E and F) Lipidomics performed in 3 NHPs per group. Lyso-GL1 level measured in plasma (E) and from 47 brain biopsy punches from 19 grey matter regions. Median with inter-quartile range across punches. ****p<0.0001, ***p<0.001; Two way-ANOVA with Tukey’s multiple comparisons test. (G – J) Histopathological analyses in NHPs with AAV.GMU01 SS3-GBA1 dose-range finding study. Histopathological findings reported with severity scores across both central nervous system and peripheral tissues by board certified clinician. Scores reported for brain (G), spinal cord (H), DRGs (I), and sciatic nerve (J). Data are Mean ± SEM. Each dot is score for individual NHP. (K) Quantification of human GBA1 protein levels in 12 brain regions from N=10 healthy human donor brain tissues (aged 55-to-75 years old) by LC-MS. Human tissue was obtained from the NIH Neurobiobank at the University of Maryland, Baltimore, MD and the Sepulveda Research Corporation. Data are Mean ± SEM. Each data point represents brain biopsy punches from each human donor. CC: corpus callosum; SN: substantia nigra; DN: dentate nucleus; Th: Thalamus, lateral nuclear group; Cb: cerebellum; PWM: periventricular white matter; Hp: hippocampus; BA: Brodmann area. (L) Quantification of human GBA1 protein levels from 3 NHPs per group. Median with inter- quartile range across 32 punches from NHPs plotted against the 12 punches from human donors. ***p<0.0001; Two way-ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Injection, Plasmid Preparation, IV Injection, Activity Assay, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: AAV GMU01-SS3-GBA1 was administered by bilateral ICV to 4-month-old Gba D409V/+ mice at 1.6e11 vector genomes per animal and 4μl per hemisphere. 6-12 animals per group. Brain, plasma, and CSF were analyzed 3-month, 6-month, and 9-month post-injection. CBE (conduritol β-epoxide) was administered at 100 mg/kg 24 hours prior to each timed necropsy. (B) Representative image of in situ hybridization to WPRE mRNA from the analyzed sagittal sections. (C – F) Vector genome assessment of the longitudinal pharmacology study with in-life duration of 3-month, 6-month, and 9-month post AAV-dosing. AAV vector genome copies (left) and Lyso- GL1 lipid clearance (right) in cortex (C), sub-cortex (D), cerebellum (E), and hindbrain (F) across all mice in the study. 6 animals for No CBE group, 12 animals for Vehicle group, and 12 animals for SS3-GBA1 group. Data are Mean ± SEM. ***p<0.0001; Two way-ANOVA with Tukey’s multiple comparisons test. (G) Lyso-GL1 lipid clearance in plasma of SS3-GBA1 injected mice. Data is Mean ± SEM. ***P<0.0001; Two way-ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Plasmid Preparation, Clinical Proteomics, Injection, In Situ Hybridization

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: 3-month-old C57/BL6 mice were injected with 4e13 VG/kg of vehicle or AAV GMU01-SS3-GBA1 intravenously. AAVs were expressed for 4 weeks followed by 100mg/kg of CBE IP injection 24 hours prior to necropsy. (B) Vector genome assessed in different visceral organs such as liver, spleen, heart, and soleus muscle. 8 animals per group. Data is Mean ± SEM. (C) Schematic view of liver sectioning strategy and representative images demonstrating robust GBA1 secretion. Vector biodistribution is shown with in situ hybridization to WPRE (top panels) and GBA1 protein expression is shown with huGBA1 immunohistochemistry after 4 weeks of expression. (D – G) Quantification of human GBA1 protein level by ELISA and Lyso-GL1 lipid clearance by LC-MS in liver (D), spleen (E), heart (F), and soleus muscle (G). 8 animals per group, No CBE group in the graphs shown is control mice that did not receive any AAV vector or CBE injection. Data are Mean ± SEM. *p<0.05, ***p<0.0001; unpaired Student’s t-test (human GBA1 protein) and ***p<0.0001; One way-ANOVA with Dunnett’s multiple comparisons test (Lyso-GL1). All groups compared to vehicle group. (H) Evaluation of vector exposure, GCase enzyme activity, and Lyso-GL1 lipid clearance from bone marrow. 8 animals per group and No CBE group in the graphs shown is control mice that did not receive any vector or CBE injection. Data are Mean ± SEM. ***p<0.0001; unpaired Student’s t-test (VGs/cell) and ***p<0.001; One way-ANOVA with Tukey’s multiple comparisons test (GCase activity and Lyso-GL1).

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Injection, Plasmid Preparation, In Situ Hybridization, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy, Control, Activity Assay

Journal:

Article Title: A novel balanced chromosomal translocation found in subjects with schizophrenia and schizotypal personality disorder: altered L-serine level associated with disruption of PSAT1 gene expression

doi: 10.1016/j.neures.2010.10.003

Figure Lengend Snippet: Chromosomal translocation and molecular profiles of the proband and her son. A) Fluorescent in situ hybridization positions the chromosome 9q21.2 breakpoint. A metaphase spread (left) and interphase nucleus (right) stained with 4',6-diamidino-2-phenylindole (DAPI) (blue). A green fluorescently labeled probe generated from RP11-45D15 BAC clone DNA is shown hybridized to the normal chromosome 9 and derived chromosomes 3 and 9 on a metaphase spread (left) stained blue with DAPI. This triple signal, also seen in an interphase nucleus (right), indicates that the DNA probe sequence spans the breakpoint. The relative proportions of signals on the two derived chromosomes suggest that the breakpoint is located towards the centromeric end of the DNA probe sequence. B) Schematic representation of the two breakpoint loci. The BAC and fosmid clones flanking the chromosome 3 breakpoint within the BC036229 transcript are shown as black rectangles. The magnified genomic context of the disruption is shown below including the location and orientation of nearby genes (nearest genes, grey block arrows: other genes, white block arrows). The chromosome 9 breakpoint does not directly disrupt a gene but is located close to PSAT1. The proximity to a segmental duplication pair (the black arrow indicates the centromeric component and the dashed line indicates the direction of the telomeric component ~1.8 Mb downstream) may indicate potential chromosome instability in this region. C) Serum level of L-serine, D-serine, and other relevant amino acids determined by HPLC. *p=0.013, Mann-Whitney U-test. Control, n=16. The open diamond indicates the proband. D) mRNA expression of the genes that are adjacent to the breakpoints. Expression level was determined by quantitative real-time RT-PCR and presented as expression relative to GAPDH mRNA. PSAT1 mRNA expression level was decreased significantly in the subjects (proband and her son) (**p=0.0053, Mann-Whitney U-test). No significant difference was observed for bobby sox homolog (Drosophila) (BBX) and centrosomal protein 78 kDa (CEP78) mRNA expression. Control: n=26 for PSAT1, n=10 for BBX and CEP78. The open diamond indicates the proband. mRNA expression of coiled-coil domain containing 54 (CCDC54), guanine nucleotide binding protein q polypeptide (GNAQ), and BC036229 was not detected in either subject or control lymphoblastoid cells. See Figure 1B for the location of each gene.

Article Snippet: Assay IDs for each target were as follows: BBX: Hs00329131_m1, CCDC54: Hs00540426_s1, CEP78: Hs00397220_m1, GNAQ Hs00387073_m1, and PSAT1: Hs00253548_m1.

Techniques: Translocation Assay, In Situ Hybridization, Staining, Labeling, Generated, Derivative Assay, Sequencing, Clone Assay, Disruption, Blocking Assay, MANN-WHITNEY, Control, Expressing, Quantitative RT-PCR, Binding Assay

Journal:

Article Title: A novel balanced chromosomal translocation found in subjects with schizophrenia and schizotypal personality disorder: altered L-serine level associated with disruption of PSAT1 gene expression

doi: 10.1016/j.neures.2010.10.003

Figure Lengend Snippet: Effects of PSAT1 knockdown by RNAi on L-serine level. A) Successful knockdown of PSAT1 by siRNA in primary rat astrocytes. Primary rat astrocytes were transfected with siRNA against PSAT1 or control siRNA. Cells were harvested on day 7 and examined for PSAT1 protein expression by Western blot analysis by using anti-PSAT1 antibody. GAPDH expression level was examined for internal control. (-); no transfection. Bar graph shows PSAT1 expression level relative to GAPDH. B) Amino acids concentration in culture supernatants of primary rat astrocytes with PSAT1 siRNA. Astrocytes transfected with either PSAT1 siRNA or control siRNA was determined at 24 h after the last medium change (day 7 post-transfection) by a high-performance liquid chromatography (HPLC) system. Error bars represent standard deviation. NT; non-treated, Con; treated with control siRNA, and Exp; treated with PSAT1 siRNA. **p=0.00175, Student t-test. Data are representative of three independent experiments.

Article Snippet: Assay IDs for each target were as follows: BBX: Hs00329131_m1, CCDC54: Hs00540426_s1, CEP78: Hs00397220_m1, GNAQ Hs00387073_m1, and PSAT1: Hs00253548_m1.

Techniques: Knockdown, Transfection, Control, Expressing, Western Blot, Concentration Assay, High Performance Liquid Chromatography, Standard Deviation

Journal: Nature Communications

Article Title: Cell state dependent effects of Bmal1 on melanoma immunity and tumorigenicity

doi: 10.1038/s41467-024-44778-2

Figure Lengend Snippet: a Upper panel : Diagram of TurboID fusion proteins. Tb: 3 Hemagglutinin (HA) tags fused to the 5’ end of TurboID; TbNLS: Nuclear localization signal fused to the 3′ end of Tb; TbWT: WT-Bmal1 fused to the 3′ end of Tb; TbdHLH: dHLH-Bmal1 fused to the 3′ end of Tb. Created with BioRender.com. Lower panel : Immunoblot for HA and Clock with whole cell lysate (Input) and proteins pulled down with HA antibody (IP: HA) from cells: KO aC3-Tb, KO aC3-TbNLS, KO aC3-TbWT and KO aC3-TbdHLH. Numbers underneath the rows represent relative expression. RE of 3. b Heatmap of peptide intensities for proteins that were biotinylated, pulled down, and digested from streptavidin beads. Enriched labeled proteins were from cells (shown in Fig. 4a) exposed to biotin and identified by LC-MS/MS analysis. No biotin treated samples were used as negative control for endogenously biotinylated proteins. BR of 2. c Peptide intensities for Clock (inset), Myh9, Actn4, and Gapdh are shown from Fig. 4b. d Immunoblot of proteins that were co-immunoprecipitated by BMAL1 antibody from nuclear extracts of cross-linked YUMM2.1 EV, YUMM2.1 WT-Bma1 and YUMM2.1 dHLH-Bmal1 cells. Normal rabbit IgG was used as antibody control; Clock and Ezh2 were separately used as positive and negative control for immunoprecipitation. RE of 3. e Immunoblot of proteins co-immunoprecipitated by Flag antibody from 293 T cells without (−) or with different Myh9 constructs (FL: Full length of Myh9 without Flag tag; Flag-tagged Myh9 Head; Flag-tagged Myh9 Tail) and Bmal1 overexpression. RE of 3. f Bmal1 and Myh9 in situ interaction in YUMM2.1 EV, WT-Bmal1 and dHLH-Bmal1 detected by Proximity Ligation Assay (PLA) using anti-Bmal1 and anti-Myh9 antibodies. Fluorescent micrographs show nuclear staining with DAPI (blue) and PLA signal (red). RE of 2. g Mean fluorescence intensity (MFI) of nuclear PLA signals from YUMM2.1 EV ( n = 66), WT-Bmal1 ( n = 43) and dHLH-Bmal1 ( n = 38) cells. Mean ± SEM. Adjust p -value by one-way ANOVA test followed by multiple comparison test. BR = biological replicate, RE = replicate experiment. Source data are provided as a Source Data file.

Article Snippet: Lysates were clarified by centrifugation at 15,000 × g at 4 °C for 15 min. To pull down biotinylated proteins, 15 μL Pierce Streptavidin Magnetic Beads (Thermo Fisher, #88816) were washed twice with 1X RIPA buffer, incubated with 400 μL clarified lysates containing 400 μg protein from each sample with rotation for 1 h at room temperature, then moved to 4 °C and incubated overnight with rotation.

Techniques: Western Blot, Expressing, Labeling, Liquid Chromatography with Mass Spectroscopy, Negative Control, Immunoprecipitation, Control, Construct, FLAG-tag, Over Expression, In Situ, Proximity Ligation Assay, Staining, Fluorescence, Comparison

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) E2012 and E2012-BPyne structures. (b) Cell membranes were photolabeled with E2012-BPyne and then clicked with TAMRA-azide and analyzed (left: Coomassie blue; right: in-gel fluorescence). (c) Biotinylated proteins were captured by E2012-BPyne and analyzed by WB. (d) IFITM3 co-immunoprecipitated with γ-secretase subunits using anti-PS1. (e) IFITM3 was co-purified with γ-secretase subunits by GY6 or 163-BP-L-biotin. (f) An interaction (red) between PS1 and IFITM3 was determined by in situ PLA in mouse primary neurons (bottom panel). Negative controls using PS1 or IFITM3 alone (top two panels), dapi (blue), F-actin (green), scale bar = 50μm. (g) Labeling of IFITM3 by E2012-Bpyne in WT and dKO MEF cells. All WB and IF images are representative of three independent experiments (except b: 2 replicates).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Fluorescence, Immunoprecipitation, Purification, In Situ, Labeling

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) LC-MS/MS analysis of the 15 kDa band identified four peptides that match with human IFITM3. (b) WB analysis of E2012-BPyne (500 nM) labeled PS1-NTF protein. (c) Structures of imidazole GSMs, acid GSM and GSIs. (d) WB analysis of E2012-BPyne labeled proteins in the absence or presence of imidazole GSMs, acid GSM and GSIs. Labeled proteins were captured and analyzed by WB for IFITM3. (e) Structures of GY6 and 163-BP-L-biotin. (f) IFITM3 co-immunoprecipitates with γ-secretase subunits. CHAPSO solubilized cell membranes were immunoprecipitated with anti-IFITM3 antibody and probed with antibodies against PS2-CTF and Pen-2. Rabbit IgG was used as a negative control. (g) IFITM3 does not co-immunoprecipitate with SPP. CHAPSO solubilized cell membranes were immunoprecipitated with a monoclonal anti-IFITM3 antibody (9D11) and probed with antibodies against SPP and IFITM3. Mouse IgG was used as a negative control. (h) Analysis of the total protein level in WT MEF or PS1/2 double KO MEF cells. The same amount of membrane proteins was loaded and analyzed by Western blotting. ( i ) IFITM3 mRNA expression levels were measured by RT-PCR in WT MEF or PS1/2 double KO MEF cells (n=6). All WB images and graphs are representative of three independent experiments (except; a/d: 2 replicates). Graphs are mean ± SD. ns, not significant, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Immunoprecipitation, Negative Control, Membrane, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) IFITM3 KD by siRNA (n=3) in HEK-APP WT cells was confirmed by WB. Scramble siRNA (SC, n=3), a negative control. (b) IFITM3 KD reduces secreted Aβ40 (n=6, ****p<0.0001) and 42 (n=6, ****p<0.0001). Aβ levels were calculated as % of SC. (c) IFITM3 was KO of U138 cells, and empty vector (EV) was used as control. IFITM3 was reintroduced by transient transfection in both EV and KO cell lines. IFITM3 expression was confirmed by WB. (d) Effect of KO and rescue of IFITM3 on γ-secretase activity for Aβ40 (n=3, *p= 0.0249, ****p<0.0001) and 42 (n=3, *p=0.0359, **p=0.0025) cleavage. (e) Comparison IC 50 of GSM E2012-BPyne against γ-secretase for Aβ40 (n=6, **p= 0.0017) and Aβ42 (n=6, ns) cleavages in U138 EV and KO cells. All WB images and graphs are representative of three independent experiments (except; e: contains data from 2 experimental replicates of n=3). Graphs are mean ± SD. Ns, not significant, two-sided Student’s t-test (except; d: one-way ANOVA and Fishers LSD test).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Negative Control, Plasmid Preparation, Control, Transfection, Expressing, Activity Assay, Comparison

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Quantification of WB showed that IFITM3 KD did not change protein expression levels of APP, Nct and PS1-NTF in HEK-APP WT cells (n=3). (b) Schematic representation of cell-free γ-secretase assay. γ-Secretase is incubated with a recombinant APP substrate in the presence of 0.25% CHAPSO. Cleaved Aβ40 and 42 species are measured with cleavage specific antibodies and AlphaLISA technology. ( c ) Schematic model showing different GSM and GSI binding sites in γ-secretase: E2012 (imidazole GSM), GSM-1 (acid GSM), and L458 (transition state analogue inhibitor, GSI). (d-f) Comparison of IC 50 of (d) GSM-25 (EV: n=9, KO: n= 8) for Aβ40 (****p<0.0001) and Aβ42 (ns), (e) GSM-1 (n=6) for Aβ40 (**p=0.0039) and Aβ42 (ns), and (f) L458 (n=3) for Aβ40 (ns) and Aβ42 (ns) cleavages in the U138 EV or KO cell lines (n≥3). (g) IFITM3 knockdown (KD) does not affect expression of γ-secretase subunits. IFITM3 was knocked-down by siRNA (6 pmol, n=3) in HEK-NotchΔE cells and scramble siRNA (SC, n=3) was used as a negative control. Cell lysates were probed by antibodies against Nct, PS1-NTF and IFITM3. β-Actin was used as a loading control. (h) Effect of IFITM3 KD on γ-secretase activity. IFITM3 KD increased γ-secretase cleaved product NICD, analyzed by WB. Cell lysates were probed by antibodies against c-myc (NotchΔE) and NICD and a representative quantification of NICD (n=8, ***p=0.001) is shown (lower panel). (i) Cell based NICD AlphaLISA assay (left panel) revealed an increase in NICD production with IFITM3 KD. Quantification of NICD (n=8, ***p=0.001) is shown in the right panel. (j) Effect of IFITM3 KO on γ-secretase activity. KO cells lines have increased γ-secretase activity as compared to the EV cell line. The NICD cleavage in vitro was measured by AlphaLISA assay (n=3,**p=0.0096). All WB images and graphs are representative of three independent experiments Graphs are mean ± SD. ns, not significant, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Incubation, Recombinant, Binding Assay, Comparison, Knockdown, Negative Control, Control, Activity Assay, In Vitro

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Protein levels of γ-secretase subunits and IFITM3 in pooled membranes from 4- and 28-month-old WT mouse brains (n=5 mice per age and sex group except n=4 for 28 female, female: **p=0.0014, male: **p=0.0065). Quantified as percent of 4-month female level. (b) γ-Secretase activity for Aβ40 (female: p=***0.0002, male: **p=0.0095) and Aβ42 (female: p=**0.0024, male: **p=0.0088) production in vitro from pooled membrane. (c) Solubilized membranes were captured by 163-BP-L-biotin and then analyzed for γ-secretase and IFITM3 levels. (d) WB for PS1-NTF and IFITM3 (left panel, representative mice shown) in membranes of 18-month-old WT (n=4) and IFITM3−/− (n=5) mouse brains. γ-Secretase activity for Aβ40 (***p=0.0004) and 42 (****p=<0.0001) cleavage (right panels). (e) APP, Nct, PS1-NTF and IFITM3 in membranes from 3- and 12-month-old WT and 5xFAD mice. Quantified as percent of 3-months-old WT (n=5 per group except n=4 for WT at 12 months)(3moWT-12mo5X: ****p<0.0001, 12moWT-12mo5X: ***p=0.004, 3mo5X-12mo5X: ***p=0.0003). (f) WB for PS1-NTF and IFITM3 (left panel) in membranes from 4-month-old 5xFAD (n=3) and IFITM3−/−; 5xFAD (n=3) mouse brains. γ-Secretase activity in vitro for Aβ40 (***p=0.0003) and 42 (**p=<0.0011) cleavage (right panels). (g) Fluorescence microscopy of amyloid plaques in cortex and hippocampus (Thioflavin-S, green) in 4-month-old PFA perfused mice (5xFAD: n=4, IFITM3−/−; 5xFAD: n=5). Scale bar = 300μm (left), 200μm (right). Number of plaques per mm 2 of tissue was calculated throughout the brain and averaged (cor (cortex): *p=0.0109, hip (hippocampus): **p=0.0026). All WB and IF images and graphs are representative of three independent experiments (except; c and quantifications in e: 2 replicates and g: every 1/6 th section throughout the brain). Bar graphs are mean ± SD. Violin plots represent median (middle line) and interquartile range (outer lines). Ns, not significant, two-sided Student’s t-test (except; e: two-way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Activity Assay, In Vitro, Membrane, Fluorescence, Microscopy

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) WBs for Nct and PS1-NTF were quantified by Odyssey imaging (n=5 mice pooled per group, except n=4 for 28F, all=ns). (b) Effect of aging on subcellular localizations of IFITM3. A hemibrain from male wild-type C57BL/6 mouse at 4 and 28 months (n=1 per group) were homogenized and layered on iodixanol gradient (2.5 – 30 %). Fractions were collected from the top and resolved by WBs for γ-secretase, IFITM3 and different subcellular markers. (c) WBs for APP, Nct, and PS1-NTF were quantified by Odyssey imaging (n=5 mice per group except n=4 for WT at 12 months). APP: 3moWT-3mo5X: ****p<0.001, 3mo5X-12mo5X: ****p<0.0001, 12moWT-12mo5X: ****p<0.0001). Nct: 3moWT-12moWT: ****p<0.001, 3mo5X-12mo5X: ****p<0.001, 12moWT-12mo5X: ****p<0.001. PS1-NTF: 3moWT-12mo5X: ***p=0.0004, 12moWT-12mo5X: ****p<0.001. ( d ) Immunostaining of IFITM3 in mouse brains. Fluorescence microscopy of IFITM3 expression in 12-month-old PFA perfused mice (WT, upper panel, 5XFAD, lower panel). Representative images of cortex, hippocampus and subiculum (left to right) show IFITM3 (green) and DAPI (blue). Scale bar = 1000μm, 200μm, 100μm (left to right). Total IFITM3 fluorescence area within the hippocampus and cortex of WT and 5XFAD was quantified using FIJI. Total IFITM3 was divided by tissue area and 5XFAD expression was normalized to average of WT (WT: n=7, 5XFAD: n=9)(cor (cortex): **p=0.0035, hip (hippocampus): ****p<0.0001 ). (e) IFITM3 expression in astrocytes and microglia is upregulated in 5XFAD mice compared to WT mice. Fluorescence microscopy of IFITM3, GFAP (top) and Iba1 (bottom) expression in 12-month-old PFA perfused mice (WT, upper panel, 5XFAD, lower panel). Representative images of the hippocampus and cortex show IFITM3 (red), GFAP (green – top), Iba1 (green - bottom), and DAPI (blue), scale bar = 500μm. Inset panels (left to right) show GFAP or Iba1 (green), IFITM3 (red) and merge. Scale bar = 50μm. All WB images and graphs are representative of three independent experiments (except; b: 2 replicates). Graphs are mean ± SD. ns, not significant, , two-sided Student’s t-test, (except; c: one-way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Imaging, Immunostaining, Fluorescence, Microscopy, Expressing

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) The expression profiles of IFITM3 in human control (n=76) and LOAD (n=80) from temporal cortex (**p=0.002)(Mayo Clinic cohort). (b) IFITM3 mRNA expression levels in control and LOAD samples (LOAD: n=18 and control: n=10, **p=0.0056). (c) The protein levels of γ-secretase subunits and IFITM3 in human brain membranes (LOAD: n=18 and control: n=10, *p=0.0127). IFITM3 quantified as relative intensity. (d) IFITM3 expression (left) between control (n=10), LOAD-L (n=10) and LOAD-H (n=8)(****p<0.0001). γ-Secretase activity for Aβ40 (**p=0.0037, ***p=0.0007) and 42 (**p=0.0036, ***p=0.0002) cleavage between groups. (e) Primary mouse neurons were treated with control (n=2), 10ng/mL (n=3, ***p=0.0005) or 100 ng/mL (n=3, ***p=0.0003) of IFN-γ, membranes were probed for γ-secretase and IFITM3 (left panel, quantified: right panel). (f) Quantification of secreted Aβ40 from 10ng/mL (n=8, **p=0.0010) and 100 ng/mL (n=8, **p=0.0026) IFN-γ treated neurons Aβ42 from 10ng/mL (n=8, ***p=0.0006) and 100 ng/mL (n=8, ***p=0.0003) IFN-γ (n=8). (g) γ-Secretase activity for Aβ40 (n=6, ***p=0.0007) and 42 (n=6, ***p=0.006) cleavage from IFN-γ treated neuron membranes. (h) Photolabeled PS-NTF in neuronal membranes and quantified as % of control (control: n=10, 10ng/ml: n=5, **p=0.0023, 100ng/ml: n=7,****p<0.0001). (i) WB for IFITM3 and PS1-NTF primary human astrocytes treated with PBS, IL-6, or IL-1β (control: n=6, IL-6: n=4, **p=0.0014, IL-1β: n=3, *p=0.0103), data normalized to PBS. (j) γ-Secretase activity for Aβ40 (control: n=8 IL-6: n=7, **p=0.0098, IL-1β: n=9,****p<0.0001) and 42 (control: n= IL-6: n=7, *p=0.0467, IL-1β: n=9, ****p<0.0001) in astrocyte membrane. All WB images and graphs are representative of three independent experiments (except; b: 1 replicate, c/g: 2 replicates, d: γ-secretase activity graph contains data from two independent replicates, h: 5 replicates). Graphs are mean ± SD. Ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Control, Activity Assay, Membrane

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Spearman’s correlation of mRNA expression of human IFITM3 gene with age was analyzed in the cortex (n=158) and hippocampus (n=123) of normal human brains using the Genotype-Tissue Expression (GTEx) cohort.. (b) mRNA expression in non-demented subject control (n=10) and LOAD samples (n=18) of MAP2 (ns), GFAP (**p=0.0046), and AIF1 (ns) were measured, which were used in – . (c) Expression profiles of MAP2 (ns), GFAP (****p<0.0001), and AIF1 (ns) in the temporal cortex of human control (n=76) and LOAD samples (n=80) using the Mayo Clinic cohort data. Correlation analyses were carried out and p values were calculated. The protein levels of Nct (****p<0.0001) and PS1-NTF (**p=0.0042) in human brain membranes (control and LOAD). The samples were analyzed by WB and quantified (n=10 and 18, respectively). Signal was normalized to HeLa cell membrane. (e-f) IFITM3 SNP Genotypes. (e) Allelic discrimination plot depicting rs34481144 genotype calls for control (n=9), LOAD-L (n=10), and LOAD-H (n=8) brain samples. The axes show delta Rn values obtained from TaqMan SNP genotyping analysis. Samples without genomic DNA were used as non-template controls (shown as black squares in the left lower quadrant, n=2). (f) Allele frequency of rs34481144 genotype in control (n=9) and LOAD (n=18). ( g ) mRNA level of IFITM3 gene in four types of EGFP/L10a-expressing mouse hippocampal neurons (GAD2 (glutamate decarboxylase 2), CCK (cholecystokinin), PV (parvalbumin), and CORT (cortistatin) expressing GABAergic neurons)(n=4 per group, GAD2-PV: ***p=0.0003, CCK-PV: ****p<0.0001, CCK-Cort *p=0.0363, PV-Cort: **p=0.0059). ( h ) mRNA levels of IFITM3 in human iPSC-derived neurons (n=4) and human primary astrocytes (n=3) were measured by qPCR (****p<0.0001). ( i-j ) Human iPSC-derived neurons (i) and human primary astrocytes (j) were stained for IFITM3 with MAP2 (neuronal marker) or S100β (astrocyte marker). DAPI was used for nucleus staining. Scale bar = 200 and 500 μm. ( k ) Induction of IFITM3 by IFN-α in primary neurons. E16 mouse primary neurons were treated with 100 ng/ml of IFN-α at DIV12 for 24 hours. The protein levels of γ-secretase and IFITM3 were analyzed by WB (n=4 per group). β-Tubulin III was used as a loading control. ( l ) Effect of IFITM3 induction on γ-secretase activity for Aβ40 (*p=0.0116) and Aβ42 (*p=0.0319) activity. Membranes from primary neurons were incubated with the recombinant APP substrate C100-ΔID-FLAG and γ-secretase activity (Aβ cleavage rate) was assayed by human Aβ three-plex MSD kits (n=12, 10). ( m ) JC8 whole cell photolabeling. Neuronal membranes were photolabeled with JC8 in the absence or presence of L458 and analyzed by anti-PS1-NTF antibody. Photolabeled PS1-NTF protein level was quantified by Odyssey imaging (n=3, *p=0.0210). (n) Spearman’s correlation between the expression level of IFITM3 and viruses. In the Brodmann Area 22 (BA-22 region) in the Mount Sinai Brain Bank (MSBB) cohort, the expression level of IFITM3 is positively correlated with the expression level of the human herpesvirus-6B (HHV-6B) (rho=0.248, p=0.044, n=66). In the Brodmann Area 36 (BA-36 region), the expression level of IFITM3 is positively correlated with the expression of hepatitis C virus genotype 4 (rho=0.255, p=0.033, n=70). All WB images and graphs are representative of two independent experiments (except; b/e: 1 replicate, h: data pooled from 2 experiments, k: 3 replicates, l: data pooled from 4 experiments). Bar graphs are mean ± SD. Violin plots represent median (middle line) and interquartile range (outer lines). ns, not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001, two-sided Student’s t-test (except; g: One-Way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Control, Membrane, Derivative Assay, Staining, Marker, Activity Assay, Incubation, Recombinant, Imaging, Virus

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Schematic model showing L458 binding to the subsites (S2-S3’) in the active site of γ-secretase. (b) Photolabeling of IFITM3 and PS1 by four inhibitors. (c) Photolabeling of IFITM3 and PS1 by L505 in human brains (control: n=4, LOAD-L: n=5, LOAD-H: n=5). (d) Pearson’s correlation between γ-secretase activity and L505 labeled IFITM3 (Fig. 5c) in LOAD samples (n=10). (e) Double cross-linking of γ-secretase and IFITM3 by the dual probe, L631. WB reveals multiple protein complex species containing IFITM3, PS1-NTF, PS1-CTF, IFITM3 homodimer, IFITM3-PS1 and PS1-NTF-CTF heterodimers. (f) Schematic representation of the interaction between IFITM3 and γ-secretase, IFITM3 is near the active site and can be crosslinked with PS1-NTF. (g) IFITM3 connects infections and innate immunity with Aβ production and AD risk. (A) Pathogenic challenge, or other inflammatory conditions, induce the release of proinflammatory cytokines from astrocytes and microglia. (B) Cytokines upregulate IFITM3 expression in neurons and astrocytes that potentiates γ-secretase, increasing Aβ production. (C) As part of an innate immune response, Aβ acts as an antimicrobial or antiviral peptide. In turn, Aβ accumulation also triggers AD pathology. All WB images and graphs are representative of three independent experiments (except; c: 2 replicates).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Binding Assay, Control, Activity Assay, Labeling, Expressing

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) Schematic representation of FLNA structure including numbering of the Ig-like domain repeats, and labelling of the actin-binding domain (ABD). The asterisks mark the repeats lacking in the FLNA variant form used in ( B ). ( B ) Pull-down assays showing direct interaction between recombinant DPP9 and recombinant FLAG tagged wt FLNA or a mutated form of FLAG-FLNA (lacking repeats 4, 9, 12, 17, 19, 21, and 23). Shown is a representative result of at least three independent experiments. ( C ) Recombinant DPP9 binds directly to GST- FLNA construct containing repeats 5–7 but not to GST-FLNA construct containing repeats 6–7. Shown is a representative result of at least three independent experiments. ( D ) Co-immunoprecipitation of endogenous FLNA with endogenous DPP9 from HeLa cells treated with different cross-linkers. Binding was observed in the presence of the sulfhydryl cross-linker DPDPB. Shown is a representative result of at least three independent experiments. To control for the specificity of the cross link, we blotted for DPP8, which did not bind to DPP9 in the presence of DPDPB ( E ) Quantification of the proximity ligation assay (in situ PLA) visualizing DPP9-FLNA interaction in HeLa cells treated with FLNA silencing oligos or non-targeting (NT) siRNAs for control shown in ( F ). The number of PLA signals per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Data are represented as mean ± SEM. Signals of more than 130 cells were quantified for each condition respectively. Statistical analysis was carried out by an unpaired two-tailed t test (***p<0.0005). ( F ) PLA showing interaction of DPP9 with FLNA in HeLa cells. Each red dot represents a single FLNA-DPP9 interaction. The number of PLA signals is significantly decreased in cells silenced for FLNA compared to cells treated with NT siRNA. Actin filaments are stained in green, nuclei were visualized by using HOECHST. Shown are representative images of at least three independent PLA experiments. DOI: http://dx.doi.org/10.7554/eLife.16370.003

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Binding Assay, Variant Assay, Recombinant, Construct, Immunoprecipitation, Control, Proximity Ligation Assay, In Situ, Software, Two Tailed Test, Staining

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) In vitro cleavage of a synthetic Syk peptide corresponding to the N-terminus of Syk (1–31) by recombinant DPP9. 50 µM of a synthetic Syk (1–31) peptide was incubated for 6 hr, either alone or with 130 nM DPP9. For control 10 µM allosteric DPP9 inhibitor SLRFLYEG was added in addition to 130 nM DPP9 and (6 hr). An additional control included the peptide and the inactive DPP9 S730G variant. Samples were analysed by high resolution liquid chromatography/tandem mass spectrometry in triplicate. Quantitation was achieved by extracting ion chromatograms and integrating peak areas for the most abundant 3+ charge state of the intact 1–31 ([M+3H] 3+ m/z 1149.8589) and the cleaved 3–31 ([M+3H] 3+ m/z 1082.4997) peptides. The identities and retention times of the peptides were established by accurate mass measurement and product ion spectra (data not shown). ( B – G ) PLA assays showing that the interaction between DPP9 and Syk requires the active site of DPP9. Shown are representative images with the corresponding quantifications of at least three independent PLA experiments. Actin filaments are stained in green, and nuclei were visualized by using HOECHST. The number of PLA signals (red dots) per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Signals of more than 300 cells were quantified for each condition respectively. Statistical analysis was carried out by an unpaired two-tailed t test (**p<0.005; ***p<0.0005; n.s = not significant). ( B ) The interaction between DPP9 and Syk is markedly decreased in HeLa cells treated with 10 µM SLRFLYEG compared to control cells treated with DMSO. ( C ) Quantification of the PLA DPP9-Syk shown in ( B ). Data are represented as mean ± SEM. ( D ) The number of PLA signals representing DPP9-Syk interactions per cell is reduced upon treatment of HeLa cells with the competitive DPP8/9 inhibitor 1G244 (10 µM, for 5 min) compared to control cells treated with DMSO. ( E ) Quantification of the PLA DPP9-Syk shown in ( D ). Data are represented as mean ± SEM. ( F ) The interaction of DPP9 with FLNA is not significantly altered upon treatment of HeLa cells with 1G244 (10 µM, 30 min) compared to control cells treated with DMSO. ( G ) Quantification of the PLA DPP9- FLNA shown in ( F ). Data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.16370.008

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: In Vitro, Recombinant, Incubation, Control, Variant Assay, Liquid Chromatography, Mass Spectrometry, Quantitation Assay, Mass Measurement, Staining, Software, Two Tailed Test

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: HeLa cells were treated with 10 µM DPP8/9 inhibitor 1G244 or DMSO for control (0, 5 and 30 min). Cells were lysed and extracts (5 µg) of were analysed for DPP activity in the presence of the artificial DPP substrate GP-AMC (250 µM) or the unrelated substrate R-AMC (50 µM). Fluorescence was measured over time. Experiment was performed at least three times, each time in triplicates. Shown is a representative, data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.16370.009

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Control, Activity Assay, Fluorescence

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) Total cell lysates (10 µg per lane) of DG-75 cells stimulated with 12 µg/ml F(ab’) 2 fragment goat-anti-human IgG+IgM (0, 1 and 4 min) were analyzed for DPP9 protein levels by Western blotting. Tubulin was used as loading control. Shown is a representative blot, an experiment was performed more than five times. ( B ) DG-75 cells were treated with 10 µM DPP8/9 inhibitor 1G244 or DMSO for control (0, 5 and 30 min). Cell lysates (10 µg) of were analysed for DPP activity in the presence of the artificial DPP substrate GP-AMC (250 µM) or the unrelated substrate R-AMC (50 µM). Fluorescence was measured over time. An experiment was performed at least three times, each time in triplicates. Shown is a representative, data are represented as mean ± SEM. ( C ) Indirect immunofluorescence images of DG-75 cells decorated with antibodies against DPP9, DPP8 and DPPIV. DOI: http://dx.doi.org/10.7554/eLife.16370.013

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Western Blot, Control, Activity Assay, Fluorescence, Immunofluorescence

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A and B ) PLA showing that the interactions of FLNA with Syk and DPP9 are conserved in human DG-75 B cells. Each PLA interaction is shown here as a white dot, nuclei were visualized by using HOECHST. Control reactions (NCtrl) were performed with only one primary antibody (αSyk, αFLNA or αDPP9). Shown are representative images and quantifications of at least three independent PLA experiments. The number of PLA signals per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Signals of more than 80 cells were quantified for each condition respectively. Data are represented as mean ± SEM. Statistical analysis was carried out by an unpaired two-tailed t test (***p<0.0001). ( C and D ) PLA in DG-75 cells showing that Syk interacts specifically with DPP9 but not with its homologs DPP8 and DPPIV. Control reactions (NCtrl) cells were treated with one primary antibody only: αDPP9, αDPP8 or αDPPIV. Shown are quantifications of the PLA DPP9-Syk, DPP8-Syk and DPPIV-Syk in DG-75 cells from three independent experiments. Data are represented as mean ± SEM. The number of PLA signals per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Signals of more than 100 cells were quantified for each condition respectively. Statistical analysis was carried out by an unpaired two-tailed t test (***p<0.0001; n.s = not significant). ( E ) CHX chase experiment showing reduced stability of endogenous Syk upon stimulation of the BCR. Human DG-75 cells were stimulated with 12 µg/ml F(ab’) 2 fragment goat-anti-human IgG+IgM (+ stim), or left untreated (- stim), and simultaneously subjected to CHX chase. DPP9 was analysed as a loading control. Shown is one representative result of at least three independent experiments. ( F ) CHX chase experiments showing that the stability of endogenous Syk in stimulated DG-75 cells, is determined by the proteasome and by DPP9. DG-75 cells were treated either with the DPP8/9 inhibitor 1G244 (10 µM), with the proteasome inhibitor MG132 (100 µM) or with DMSO for control (MOCK). Cell lysates were analysed for protein levels of Syk and of DPP9 for loading control by Western blotting. Shown is one representative result of at least three independent experiments. ( G ) Quantification of the Western blot results shown in ( F ). The ratio of Syk/DPP9 at time 0 hr was normalized to 100%. For signal quantification GelQuant.NET software provided by biochemlabsolutions.com was used. ( H ) CHX chase experiment assaying the stability of endogenous phosphorylated Syk (p-Y323) in stimulated DG-75 cells upon treatment with the DPP8/9 inhibitor 1G244 (10 µM) or with DMSO for control (MOCK). Tubulin was assayed as loading control. Shown is one representative result of at least three independent experiments. ( I ) Quantification of the Western blot results shown in ( H ). The ratio of Syk p-Y323/tubulin at time 10 min was normalized to 100%. For signal quantification GelQuant.NET software provided by biochemlabsolutions.com was used. DOI: http://dx.doi.org/10.7554/eLife.16370.012

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Control, Software, Two Tailed Test, Western Blot

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) Higher levels of endogenous active Syk (phosphorylated on Y352) in stimulated DG-75 cells treated with the DPP8/9 inhibitor 1G244 compared to the mock (DMSO) treated cells. 1G244 (10 µM) was added at the same time of BCR stimulation (time 0). Tubulin was assayed for loading control. Shown is a representative result of at least three independent pulse chase experiments. ( B ) Quantification of the Western blot results shown in ( A ). The ratio of Syk p-Y352/tubulin at time 10 min was normalized to 100%. For signal quantification GelQuant.NET software provided by biochemlabsolutions.com was used. ( C ) Inhibition of DPP9 in DG-75 cells leads to increased Ca 2+ mobilization, which is not dependant on BCR stimulation. Shown are flow cytometric Ca 2+ profiles after the addition of 10 µM DPP8/9 inhibitor 1G244 or DMSO for control (marked by an arrow). To monitor Ca 2+ mobilization upon BCR stimulation in either 1G244-treated or control cells, cells were treated with 10 µg/ml F(ab) 2 goat-anti-human IgM. ( D ) Same as in ( C ) using Ramos cells as a second B cell line. ( E – F ) In the absence of BCR stimulation, DPP9 inhibition leads to higher basal levels of phosphorylated Syk and its down stream effector protein PLCγ2. ( E ) Western blotting analysis of DG-75 cells treated with 1G244 in the absence of BCR stimulation. Lysates were analysed with antibodies specific against phosphorylated Syk (p-Y352) or phosphorylated PLCγ2. For loading control lysates were analysed with antibodies recognizing unmodified Syk and PLCγ2. ( F ) Cells were treated for 20 min with 1G244 (10 µM) or DMSO for control. Alternatively, cells were treated for 30 min with the allosteric DPP9 inhibitor SLRFLYEG peptide complexed with the carrier peptide (pep-1). Control cells were treated with the carrier peptide only. Following inhibitor treatment, cells were lysed and subjected to immunoprecipitation assays against Phospho-Y. Eluted proteins were analysed for Syk and PLCγ2 levels by Western blotting. Total protein levels in cell lysates were monitored for control. ( G ) Lower levels of phosphorylated ERK1/2 (both bands) are detected in the 1G244 treated DG-75 cells compared to mock (DMSO) treated cells. 1G244 (10 µM) was added prior to BCR stimulation (time 0). DPP9 was assayed for loading control. Shown is a representative result of at least three independent pulse chase experiments. ( H ) Quantification of the Western blot results shown in ( G ) as described in ( B ). DOI: http://dx.doi.org/10.7554/eLife.16370.015

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Control, Pulse Chase, Western Blot, Software, Inhibition, Immunoprecipitation

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-C: Bacterially purified GST-Bub2 or GST-Bub2-Q132L, MBP-Bfa1 and 6xHis-Tem1 proteins were used to measure the kinetics of hydrolysis+dissociation (γ[ 32 P]GTP) or dissociation only (γ[ 35 S]GTP) using a filter binding assay (see ). Graphs show average values and standard deviations from three independent experiments. D: Exponentially growing cultures of the indicated strains were shifted to nocodazole containing medium at t = 0. Cell samples were withdrawn at the indicated time for FACS analysis of DNA contents. E: The percentage of cells with binucleate cell bodies accompanied or not by a checkpoint defect (indicated by re-budding in the absence of proper chromosome segregation) was scored in cycling cultures of the indicated strains shifted either to 14°C for 16h (left graph) or to 37°C for 3h (right graph). F-G: Exponentially growing cells with the indicated genotypes were arrested in G1 by α-factor and released into fresh medium at time 0. At 70’ after release α-factor was re-added to prevent cells from entering a second cell cycle. Cell samples were collected for FACS analysis of DNA contents (F) and for tubulin staining by indirect immunofluorescence (G). H: Cells were treated as in (F-G). TCA extracts were prepared from cell samples at the indicated time points to monitor kinetics of Bfa1-HA6 phosphorylation and Clb2 accumulation and degradation by western blot analysis. Pgk1 was used as loading control. I: Protein extracts from cells expressing the indicated tagged proteins were used for immunoprecipitation with an anti-HA affinity resin. Western blot analysis was then performed with anti-GFP and anti-HA antibodies. The input represents 1/25 th of the total extract used for each IP. J-K: Localization of eGFP- tagged Bub2/Bub2-Q132L, Tem1, Bfa1 (J) and Cdc15-GFP (K) was analysed by fluorescence microscopy after formaldehyde fixation.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Purification, Filter-binding Assay, Staining, Immunofluorescence, Western Blot, Expressing, Immunoprecipitation, Fluorescence, Microscopy

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Cycling cells co-expressing Spc72-Bfa1-eGFPand Spc42-mCherry to mark the SPB (upper panel) or co-expressing Tem1-eGFP and Tub1-GFP (to mark microtubules, lower panel) were analysed to study the distribution of Spc72-Bfa1-eGFP (A) and Tem1-eGFP (B) at SPBs in SPC72-BFA1 bfa1Δ cells. C-D: Cycling cells with the indicated genotypes were shifted into nocodazole containing medium (t = 0). Cell samples were withdrawn at the indicated times for FACS analysis of DNA contents. E: The percentage of cells with binucleate cell bodies accompanied or not by a SPOC defect was scored after propidium iodide staining of cycling cultures of cells with the indicated genotypes after shift to 14°C for 16h. The histograms on the right side represent the DNA contents of the same cells as measured by FACS analysis. F: Percentage of metaphase cells with Cdc15-GFP at 0, 1 or 2 SPBs was scored in the indicated strains after formaldehyde fixation. Metaphases were identified by means of the Tub1-mCherry co-expressed marker. G: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YPD and incubated at the indicated temperature. H: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YP medium containing either glucose or galactose and incubated at 25°C for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Expressing, Staining, Marker, Incubation

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A: Bacterially purified 6XHis-Tem1 and 6XHis-Tem1-Q79L were loaded with γ[ 32 P]GTP either in the absence or in the presence of recombinant MBP-Bfa1 and incubated at 30°C for 10 minutes. The mixture was then added to GST-Bub2 or buffer alone and kinetics of GTP hydrolysis and dissociation was followed by a filter-binding assay (see details in ). Graphs show average values and standard deviations from three independent experiments. B: Wild type and TEM1-Q79L cells were arrested in G1 by α-factor and then released into fresh medium at 25°C (t = 0). Cell samples were withdrawn every 10’ to measure kinetics of budding and spindle formation/elongation after in situ immunostaining of tubulin. C: Actomyosin ring contraction has been visualized by live cell imaging of wild type and TEM1-Q79L expressing Myo1-GFP (n = 30). D: Logarithmically growing cultures of cells with the indicated genotypes were shifted into nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. E: The percentage of cells with binucleate cell bodies accompanied or not by SPOC defect was scored after DAPI staining of cycling cells of the indicated strains shifted to 14°C for 16h. F: Logarithmically growing cultures of strains with the indicated genotypes were shifted to nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. G: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YPD or YP galactose plates and incubated at 30°C for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Purification, Recombinant, Incubation, Filter-binding Assay, In Situ, Immunostaining, Live Cell Imaging, Expressing, Flow Cytometry, Staining

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: List of non-essential genes implicated in microtubules dynamics or spindle positioning identified in the SGA screen with TEM1-Q79L .

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Migration

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Protein extracts from cells expressing the indicated tagged proteins were used for immunoprecipitation with an anti-HA affinity resin. Western blot analysis was then performed with anti-PK, anti-GFP and anti-HA antibodies. The input represents 1/25 th of the total extract used for each IP. C-F: Localization of eGFP- tagged Tem1 and Tem1-Q79L (C-D) or Bfa1-eGFP (E-F) was analysed in the indicated strains by fluorescence microscopy after formaldehyde fixation. Metaphase and anaphase cells were identified by means of the Tub1-mCherry co-expressed marker. Micrographs show representative cells of each strain in anaphase. G: Fluorescence intensity ratios were calculated between the two SPBs in anaphase cells of the indicated strains (see details in ).

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Expressing, Immunoprecipitation, Western Blot, Fluorescence, Microscopy, Marker

Journal: PLoS Genetics

Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

doi: 10.1371/journal.pgen.1004938

Figure Lengend Snippet: A-B: Distribution of Cdc15-GFP (A) or Mob1-GFP (B) was analysed in the indicated strains by fluorescence microscopy after formaldehyde fixation. Metaphase and anaphase cells were identified by means of the Tub1-mCherry co-expressed marker. Micrographs show representative wild type and TEM1-Q79L cells expressing Cdc15-GFP in metaphase. C: Serial dilutions of stationary phase cells with the indicated genotypes were spotted on YPD and incubated at the indicated temperatures for 48h.

Article Snippet: Proteins transferred to Protran membranes (Schleicher & Schuell) were probed with anti-PK mouse monoclonal antibodies for PK-tagged Bub2, with anti-GFP rat monoclonal antibodies for GFP-tagged Tem1, Bub2 and Bfa1 (Chromotek) and with an anti-HA monoclonal antibody (12CA5).

Techniques: Fluorescence, Microscopy, Marker, Expressing, Incubation