Journal: Antiviral Research

Article Title: Triggering unfolded protein response by 2-Deoxy- d -glucose inhibits porcine epidemic diarrhea virus propagation

doi: 10.1016/j.antiviral.2014.03.007

Figure Lengend Snippet: 2-DG treatment affects virus packaging. (A) Vero cells were pretreated with 10 mM 2-DG for 24 h, and the cells were analyzed by FACS analysis using anti-CD13 polyclonal antibody. (B, C) 2-DG treatment did not affect virus entry as determined by RT-PCR and Western blot analysis. For RT-PCR analysis, the cells were treated with 10 mM 2-DG before virus infection. Bars represent mean percent copy number of viral RNA levels in cells by RT-PCR using primers specific for PEDV N gene. Cell lysates were analyzed by Western blot using antibody against PEDV N. (D) The Vero cells were infected with PEDV for 2 h (MOI = 1), 2-DG (10 mM) was incubated with the cells. The cells were harvested at different time points. Cell lysates were analyzed by Western blot using antibody against PEDV N. (E, F) The Vero cells was treated with different concentrations 2-DG after infected with PEDV (MOI = 0.01) for 2 h, the ratio of PEDV RNA copy numbers between the supernatants and the cell lysates was detected by Q-PCR, and the ratio of the virus titers in supernatants and cell lysates were determined by plaque assay.

Article Snippet: Detached Vero cells were washed once with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and stained with rabbit anti-human CD13 protein (Boster Bio-Tech Co. Ltd., Wuhan, China) for 1 h at 4 °C.

Techniques: Virus, Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Incubation, Plaque Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Blood–spinal cord barrier pericyte reductions contribute to increased capillary permeability

doi: 10.1038/jcbfm.2012.113

Figure Lengend Snippet: Pericyte number and coverage is reduced along blood–spinal cord barrier. ( A ) Confocal microscopy analysis of CD13-positive pericytes (green) and collagen IV-positive capillary profiles (red) in 2-month-old wild-type mouse cortex, caudate, and hippocampal brain regions and cervical, thoracic, and lumbar spinal cord regions (anterior horns). ( B ) Quantification of regional CD13-positive pericyte coverage of collagen IV-positive brain and spinal cord capillaries. Mean±s.e.m., n =5 to 7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05. ( C ) Quantification of regional CD13-positive pericyte cell number normalized to collagen IV-positive capillary surface area in the brain and spinal cord. Mean±s.e.m., n =5 to 7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05. ( D ) Confocal microscopy analysis of platelet-derived growth factor receptor β (PDGFR β )-positive pericytes (green) and lectin capillary profiles (red) in 2-month-old wild-type mouse cortex, caudate, and hippocampal brain regions and cervical, thoracic, and lumbar spinal cord regions. ( E ) Quantification of regional PDGFR β -positive pericyte coverage of lectin-positive brain and spinal cord capillaries. Mean±s.e.m., n =7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05. ( F ) Quantification of regional PDGFR β -positive pericyte cell number normalized to lectin-positive capillary surface area in the brain and spinal cord. Mean±s.e.m., n =7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05.

Article Snippet: Sections were then incubated in the following primary antibodies: goat α -mouse CD13 (10 μ g/mL, R&D Systems), mouse α -mouse desmin (1:100, Dako, Carpinteria, CA, USA), rabbit α -mouse collagen IV (1:200, EMD Millipore), rabbit α -human fibrin which cross reacts with mouse fibrin (1:500, Dako), goat α -mouse thrombin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse α -mouse zonula occludens-1 (ZO-1) (1:50, Invitrogen), mouse α -mouse occludin (1:50, Invitrogen), mouse α -mouse neuronal-specific antigen A60 (NeuN) (1:100, EMD Millipore) and goat anti-mouse ChAT (choline acetyl transferase) (1:50, EMD Millipore).

Techniques: Confocal Microscopy, Derivative Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Blood–spinal cord barrier pericyte reductions contribute to increased capillary permeability

doi: 10.1038/jcbfm.2012.113

Figure Lengend Snippet: Subregional variation in spinal cord pericyte number and coverage between anterior horn gray matter and lateral and dorsal funiculi. ( A ) Confocal microscopy analysis of CD13-positive pericytes (green) and collagen IV-positive capillary profiles (red) in 2-month-old wild-type mouse anterior horn gray matter or dorsal columns for cervical thoracic and lumbar spinal cord. ( B ) Quantification of CD13-positive pericyte coverage of collagen IV-positive capillaries in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =5 to 7 animals per group; * P <0.05. ( C ) Quantification of CD13-positive pericyte cell number normalized to lectin-positive capillary surface area in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =5 to 7 animals per group; * P <0.05. ( D ) Quantification of platelet-derived growth factor receptor β (PDGFR β )-positive pericyte coverage of lectin-positive capillaries in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =7 animals per group; * P <0.05. ( E ) Quantification of PDGFR β -positive pericyte cell number normalized to lectin-positive capillary surface area in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =7 animals per group; * P <0.05.

Article Snippet: Sections were then incubated in the following primary antibodies: goat α -mouse CD13 (10 μ g/mL, R&D Systems), mouse α -mouse desmin (1:100, Dako, Carpinteria, CA, USA), rabbit α -mouse collagen IV (1:200, EMD Millipore), rabbit α -human fibrin which cross reacts with mouse fibrin (1:500, Dako), goat α -mouse thrombin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse α -mouse zonula occludens-1 (ZO-1) (1:50, Invitrogen), mouse α -mouse occludin (1:50, Invitrogen), mouse α -mouse neuronal-specific antigen A60 (NeuN) (1:100, EMD Millipore) and goat anti-mouse ChAT (choline acetyl transferase) (1:50, EMD Millipore).

Techniques: Confocal Microscopy, Derivative Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Blood–spinal cord barrier pericyte reductions contribute to increased capillary permeability

doi: 10.1038/jcbfm.2012.113

Figure Lengend Snippet: Exacerbation of pericyte deficiency leads to overt blood–spinal cord barrier disruption and leakage of endogenous plasma proteins. ( A ) Confocal microscopy analysis of CD13-positive pericytes (green) and collagen IV-positive capillary profiles (red) in 6-month-old Pdgfrβ +/+ and Pdgfrβ F7/F7 mouse cervical, thoracic, and lumbar spinal cord anterior horn. ( B ) Quantification of regional CD13-positive pericyte coverage of collagen IV-positive anterior horn spinal cord capillaries. Mean±s.e.m., n =3 animals per group; * P <0.05. ( C ) Representative confocal microscopy analysis of IgG (green), fibrin (red), and lectin-positive capillaries (blue) in 6-month-old Pdgfrβ +/+ mouse lumbar anterior horn and anterior horn from Pdgfrβ F7/F7 cervical, thoracic, and lumbar spinal cord. ( D , E ) Quantification of IgG ( D ) and fibrin ( E ) extravascular deposits in the spinal cord regions in tissue sections from 2-month-old B6SJL/F1 Pdgfrβ +/+ , 6-month-old Pdgfrβ +/+ 129S1/SvlmJ, and 6-month-old Pdgfrβ F7/F7 mice. Mean±s.e.m., n =3 animals per group; * P <0.05. ( F , G ) Confocal microscopy analysis of thrombin (white) ( F ) or fibrin (red) ( G ) and SMI-311-positive neurons (blue) in 6-month-old Pdgfrβ +/+ and Pdgfrβ F7/F7 lumbar spinal cord. Arrows indicate neuronal accumulation.

Article Snippet: Sections were then incubated in the following primary antibodies: goat α -mouse CD13 (10 μ g/mL, R&D Systems), mouse α -mouse desmin (1:100, Dako, Carpinteria, CA, USA), rabbit α -mouse collagen IV (1:200, EMD Millipore), rabbit α -human fibrin which cross reacts with mouse fibrin (1:500, Dako), goat α -mouse thrombin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse α -mouse zonula occludens-1 (ZO-1) (1:50, Invitrogen), mouse α -mouse occludin (1:50, Invitrogen), mouse α -mouse neuronal-specific antigen A60 (NeuN) (1:100, EMD Millipore) and goat anti-mouse ChAT (choline acetyl transferase) (1:50, EMD Millipore).

Techniques: Disruption, Clinical Proteomics, Confocal Microscopy

Journal: Journal of neuropathology and experimental neurology

Article Title: Cerebrospinal Fluid Particles in Alzheimer Disease and Parkinson Disease

doi: 10.1097/NEN.0000000000000207

Figure Lengend Snippet: Measurement of cerebrospinal fluid (CSF) particles using Apogee A50 flow cytometry. (A) Calibration Beadmix was used to assess light scatter and fluorescence. Shown are typical data from the BeadMix analyzed on the A50-Micro flow cytometer. The left cytogram shows that all populations included, 2 green fluorescent (488 nm laser) polystyrene beads with diameters of 0.11 μm and 0.5 μm, and 6 silica beads with diameters of 0.18, 0.24, 0.30, 0.59, 0.88 and 1.30 μm, were resolved from each other and from instrument noise. The right cytogram was gated to exclude the fluorescent latex beads so that the non-fluorescent beads can be evaluated more clearly. (B) Histograms show side scattering (left) and forward scattering (middle) from 0.18, 0.24, 0.30, 0.59, 0.88 and 1.3 μm silica microspheres (blue peaks) and the noise threshold limit (dotted line). Representative dot plot (right) of a CSF sample showing “smaller” particle gate based on forward angle light scatter from 0.30 μm silica microspheres and “larger” particle gate based on forward angle light scatter from 0.30 to 0.59 μm silica microspheres. (C) Flow cytometric analysis of CSF apolipoprotein-bearing particles in human CSF. Fluorescence analysis of CSF samples labeled with apoE-Alexa Fluor 488, apoJ-Alexa Fluor 488, or apoAI-FITC (right panels). Density plots show background from phosphate-buffered saline (PBS) labeled with the same antibodies (left panels) used as background control. Isotype control of corresponding species of IgG was used to label the same CSF samples (middle panels) as negative controls and results used to set gating for positive events. (D) Fluorescence analysis of a CSF sample labeled with annexin V-FITC with binding buffer containing Ca2+ (right). The same labeling procedure but using buffer without Ca2+ was used as negative control (middle). PBS labeled with annexin V-FITC was used as background control (left). (E) Flow cytometric analysis of β-amyloid (Aβ42)-positive particles in human CSF. PBS (upper, left) and isotype control (upper, right) was used as controls. A representative fluorescence plot shows Aβ42-positive events in CSF (lower, left) that were nearly completely removed following pull down of Aβ42 with 6E10 antibody coupled to agarose beads (lower, right). (F) The protein retrieved from the 6E10 pull down pellet was analyzed by Western blot using 6E10 (right) or Aβ42 (left) antibody to reveal expected smear of Aβ42-immunoreactive molecular masses (and immunoglobulin bands). PBS was substituted for CSF as the negative control.

Article Snippet: The following antibodies were employed: rabbit anti-Aβ 1-42 monoclonal antibody (clone H31L21, Invitrogen, Carlsbad, CA), which recognizes human and mouse Aβ 1-42 ; cross reactivity to Aβ 1-40 is not observed in sandwich ELISA according to the manufacturer’s instructions; mouse anti-human Aβ monoclonal antibody (clone 6E10, Covance, Princeton, NJ), mouse anti-human apoE monoclonal antibody (clone 1H4, Abcam, Cambridge, MA); mouse anti-human apoJ monoclonal antibody (clone 3R3/2, Lifespan Biosciences, Seattle, WA); rabbit anti-mouse apoJ polyclonal antibody (Lifespan Biosciences); fluorescein isothiocyanate (FITC)-conjugated mouse anti-human apoAI monoclonal antibody (clone APO-1-1, Fitzgerald Industries, Acton, MA); rabbit anti-mouse apoAI polyclonal antibody (Lifespan Biosciences); FITC-conjugated annexin V (Beckman Coulter, Pasadena, CA); and mouse anti-human tau monoclonal antibody (clone TAU-5, Abcam).

Techniques: Flow Cytometry, Fluorescence, Labeling, Binding Assay, Negative Control, Western Blot

Journal: Journal of neuropathology and experimental neurology

Article Title: Cerebrospinal Fluid Particles in Alzheimer Disease and Parkinson Disease

doi: 10.1097/NEN.0000000000000207

Figure Lengend Snippet: Cerebrospinal fluid (CSF) particles were classified by the presence of apoE, apoJ, apoAI, or β-amyloid (Aβ42), as detected by labeled antibodies or by binding of annexin V, and stratified by size into larger (A, 300 to 590 nm) or smaller (B, < 300 nm) particles for each sample. One-way ANOVA for each molecularly-defined particle concentration (events/μL) in these 2 size ranges each had p < 0.0001. Corrected multiple comparisons for CSF particle concentration had p < 0.01 for all paired comparisons except for apoE-positive vs. apoJ-positive, which were not significantly different for either size range.

Article Snippet: The following antibodies were employed: rabbit anti-Aβ 1-42 monoclonal antibody (clone H31L21, Invitrogen, Carlsbad, CA), which recognizes human and mouse Aβ 1-42 ; cross reactivity to Aβ 1-40 is not observed in sandwich ELISA according to the manufacturer’s instructions; mouse anti-human Aβ monoclonal antibody (clone 6E10, Covance, Princeton, NJ), mouse anti-human apoE monoclonal antibody (clone 1H4, Abcam, Cambridge, MA); mouse anti-human apoJ monoclonal antibody (clone 3R3/2, Lifespan Biosciences, Seattle, WA); rabbit anti-mouse apoJ polyclonal antibody (Lifespan Biosciences); fluorescein isothiocyanate (FITC)-conjugated mouse anti-human apoAI monoclonal antibody (clone APO-1-1, Fitzgerald Industries, Acton, MA); rabbit anti-mouse apoAI polyclonal antibody (Lifespan Biosciences); FITC-conjugated annexin V (Beckman Coulter, Pasadena, CA); and mouse anti-human tau monoclonal antibody (clone TAU-5, Abcam).

Techniques: Labeling, Binding Assay, Concentration Assay

Journal: Journal of neuropathology and experimental neurology

Article Title: Cerebrospinal Fluid Particles in Alzheimer Disease and Parkinson Disease

doi: 10.1097/NEN.0000000000000207

Figure Lengend Snippet: Flow cytometric analysis of the association of β-amyloid (Aβ42) with other cerebrospinal fluid (CSF) particles. (A) Dual fluorescence analysis of a representative CSF sample showing Aβ42 co-labeled with apoE-positive (upper left) or apoJ-positive (upper right) particles but not apoAI-positive (lower left) or annexin V-positive (lower right) particles. (B) Scatter plot with best-fit line for apoE associated Aβ42 particles vs. apoJ associated Aβ42 particles for all 131 participants.

Article Snippet: The following antibodies were employed: rabbit anti-Aβ 1-42 monoclonal antibody (clone H31L21, Invitrogen, Carlsbad, CA), which recognizes human and mouse Aβ 1-42 ; cross reactivity to Aβ 1-40 is not observed in sandwich ELISA according to the manufacturer’s instructions; mouse anti-human Aβ monoclonal antibody (clone 6E10, Covance, Princeton, NJ), mouse anti-human apoE monoclonal antibody (clone 1H4, Abcam, Cambridge, MA); mouse anti-human apoJ monoclonal antibody (clone 3R3/2, Lifespan Biosciences, Seattle, WA); rabbit anti-mouse apoJ polyclonal antibody (Lifespan Biosciences); fluorescein isothiocyanate (FITC)-conjugated mouse anti-human apoAI monoclonal antibody (clone APO-1-1, Fitzgerald Industries, Acton, MA); rabbit anti-mouse apoAI polyclonal antibody (Lifespan Biosciences); FITC-conjugated annexin V (Beckman Coulter, Pasadena, CA); and mouse anti-human tau monoclonal antibody (clone TAU-5, Abcam).

Techniques: Fluorescence, Labeling

Journal:

Article Title:

doi:

Figure Lengend Snippet: RAGE and HMGB1 expression in normal control (NC) and in Graves' ophthalmopathy (GO) tissues. ( a) Statistical analysis of RAGE and HMGB1 expression in GO tissues compared to NC. The sections were scored as described in Material and Methods. (b) Representative images of immunohistochemistry staining. B1: isotype negative control of GO tissue. B2: positive control. (a) RAGE expression on head and neck squamous cell carcinoma tissue. (b) HMGB1 expression on nasal cavity mucosa. B3: RAGE expression in GO tissue. B4: RAGE expression in NC tissue. B5: HMGB1 expression in GO tissue (arrows). B6: HMGB1 expression in NC tissue (arrows). B7: hematoxylin and eosin (H+E) staining of GO tissue. B8: H+E staining of NC tissue. p value ≤ 0.05 was considered to be significant. Bar = 150 μ m.

Article Snippet: Paraffin sections of GO patients and NC patients were immunostained using NovoLink Polymer Detection Systems (Novocastra Laboratories, Newcastle, UK) and the following primary antibodies diluted 1 : 100 in Antibody Diluent (Dako): rabbit polyclonal anti-human HMGB1 (LS-C2691, LifeSpan BioSciences, Inc., Seattle, WA, USA) and mouse monoclonal anti-human RAGE (LS-B6042, LifeSpan BioSciences, Inc., Seattle, WA, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Negative Control, Positive Control

Journal:

Article Title:

doi:

Figure Lengend Snippet: RAGE and HMGB1 expression on inflammatory infiltrates in GO tissues. (a) Isotype negative control staining of GO tissue. (b) RAGE expression on inflammatory infiltrates (arrows). (c) HMGB1 expression on inflammatory infiltrates (arrows). (d) H+E staining of GO tissue. Bar = 150 μ m.

Article Snippet: Paraffin sections of GO patients and NC patients were immunostained using NovoLink Polymer Detection Systems (Novocastra Laboratories, Newcastle, UK) and the following primary antibodies diluted 1 : 100 in Antibody Diluent (Dako): rabbit polyclonal anti-human HMGB1 (LS-C2691, LifeSpan BioSciences, Inc., Seattle, WA, USA) and mouse monoclonal anti-human RAGE (LS-B6042, LifeSpan BioSciences, Inc., Seattle, WA, USA).

Techniques: Expressing, Negative Control, Staining

Journal: Cell reports methods

Article Title: Identification of activity-based biomarkers for early-stage pancreatic tumors in blood using single-molecule enzyme activity screening.

doi: 10.1016/j.crmeth.2023.100688

Figure Lengend Snippet: Figure 1. Development of PMAC as a core scaffold for the microdevice-based assay (A) Concept of single-molecule enzyme activity analysis using microfabricated chambered device. (B) The design of the microfabricated chambered device for single-molecule enzyme activity analysis. (C) Structure of PMAC-based fluorogenic probes of amidases, peptidases, and proteases. (D) Absorbance spectra of EP-PMAC (5 mM) before (black line) and after (red line) a reaction with recombinant DPP4 (300 ng/mL) in Tris-HCl buffer (pH 7.4, 100 mM) containing CHAPS (0.1%) at 25C. (E) Fluorescence spectra of EP-PMAC in the same condition as in (d). Fluorescence spectra were measured every 10 min. lex. = 350 nm. (F) Structures of GP-AMC and EP-PMAC and their kinetic parameters in the reaction with DPP4 in 384-well plate-based assay. (G) Fluorescence images of microdevice loaded with GP-PMAC or EP-PMAC (50 mM) and recombinant DPP4 (10 ng/mL) in HEPES buffer (pH 7.4, 100 mM) containing CaCl2 (1 mM), MgCl2 (1 mM), DTT (1 mM), and Triton X-100 (3 mM). Images were taken after incubation at 37C for 2 h.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-DPP4 (CD26) mouse monoclonal antibody Origene Cat. #TA500733 Lot #F002; RRID: AB_11140529 Anti-CD13 rabbit monoclonal antibody Abcam Cat. #ab108310 Lot #GR3306688-9; RRID:AB_10866195 Anti-mouse IgG HRP-linked antibody Cytiva Cat. #NA931V Lot #17479274; RRID: AB_772210 Anti-rabbit IgG HRP-linked F(ab’)2 fragment Cytiva Cat. #NA9340V Lot #17402176; RRID: AB_772191 Chemicals, peptides, and recombinant proteins DPP4, human recombinant R&D Systems Cat. #9168-SE Lot #DFQM0420081 Aminopeptidase N, human recombinant R&D Systems Cat. #3815-ZN Lot #PQP0221041 Bovine serum albumin Wako Cat. #016-15091 Lot #LEQ2235 FC-70 Sigma-Aldrich Cat. #F9880 Fomblin PFPE Solvay Cat. #YL VAC 25/6 Bestatin Tokyo Chemical Industries Cat. #U0111 Saxagliptin Tokyo Chemical Industries Cat. #A3435 NHS-activated Sepharose 4 Fast Flow Cytiva Cat. #17-0906-01 Critical commercial assays IEF gels (pH 3–7) Invitrogen Cat. #EC6645BOX Bis-Tris Native PAGE gels (4–16%) Invitrogen Cat. #BN1004BOX Native Mark unstained protein standard Invitrogen Cat. #LC0725 Sainome diced electrophoresis gel assay plate Sainome Corp. Cat. #SAI-386-PEB HDL purification kit (ultracentrifugation free) Cell Biolabs Inc. Cat. #STA-607 Bio-Rad protein assay dye reagent concentrate Bio-Rad Cat. #5000006JA

Techniques: Activity Assay, Recombinant, Fluorescence, Incubation

Journal: Cell reports methods

Article Title: Identification of activity-based biomarkers for early-stage pancreatic tumors in blood using single-molecule enzyme activity screening.

doi: 10.1016/j.crmeth.2023.100688

Figure Lengend Snippet: Figure 6. Identification of high- and low-activity species of single-molecule DPP4 and its alteration in blood samples of patients with pancreatic tumors (A) Fluorescence images of microdevice loaded with EP-PMAC (50 mM) and plasma samples (1:5,000 dilution; top) or recombinant DPP4 (1 ng/mL; bottom). The histograms indicate the activity distribution of single-molecule enzymes. White arrows indicate the low-activity DPP4 species detected in plasma. (B) Histograms of single-molecular activities of EP-PMAC (50 mM) with blood samples (1:5,000 dilution) of patients with early-stage pancreatic tumor (stages I and II, 30 individuals) and healthy subjects (30 individuals). Samples with aberrant ratio of high- and low-activity species of DPP4 were indicated by red square. (C) Left: dotted plot of the proportion of high-activity DPP4 species in plasma of early-stage (stages I and II) pancreatic tumor patients or of healthy subjects in (B). p value was calculated using Student’s t test. Right: dot plot of the total DPP4 activity in plasma samples monitored using conventional 384-well plate-based fluorometric assay. EP-PMAC (10 mM) was incubated with plasma samples (1:500 dilution) in HEPES buffer (pH 7.4, 100 mM) containing CaCl2 (1 mM), MgCl2 (1 mM), DTT (1 mM), and CHAPS (0.1%) at 25C for 30 min. p value was calculated using Student’s t test. (D) Western blot analysis of DPP4 of plasma samples of patients with pancreatic tumors that showed altered DPP4 populations or that of healthy subjects.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-DPP4 (CD26) mouse monoclonal antibody Origene Cat. #TA500733 Lot #F002; RRID: AB_11140529 Anti-CD13 rabbit monoclonal antibody Abcam Cat. #ab108310 Lot #GR3306688-9; RRID:AB_10866195 Anti-mouse IgG HRP-linked antibody Cytiva Cat. #NA931V Lot #17479274; RRID: AB_772210 Anti-rabbit IgG HRP-linked F(ab’)2 fragment Cytiva Cat. #NA9340V Lot #17402176; RRID: AB_772191 Chemicals, peptides, and recombinant proteins DPP4, human recombinant R&D Systems Cat. #9168-SE Lot #DFQM0420081 Aminopeptidase N, human recombinant R&D Systems Cat. #3815-ZN Lot #PQP0221041 Bovine serum albumin Wako Cat. #016-15091 Lot #LEQ2235 FC-70 Sigma-Aldrich Cat. #F9880 Fomblin PFPE Solvay Cat. #YL VAC 25/6 Bestatin Tokyo Chemical Industries Cat. #U0111 Saxagliptin Tokyo Chemical Industries Cat. #A3435 NHS-activated Sepharose 4 Fast Flow Cytiva Cat. #17-0906-01 Critical commercial assays IEF gels (pH 3–7) Invitrogen Cat. #EC6645BOX Bis-Tris Native PAGE gels (4–16%) Invitrogen Cat. #BN1004BOX Native Mark unstained protein standard Invitrogen Cat. #LC0725 Sainome diced electrophoresis gel assay plate Sainome Corp. Cat. #SAI-386-PEB HDL purification kit (ultracentrifugation free) Cell Biolabs Inc. Cat. #STA-607 Bio-Rad protein assay dye reagent concentrate Bio-Rad Cat. #5000006JA

Techniques: Activity Assay, Fluorescence, Clinical Proteomics, Recombinant, Incubation, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: Interaction of peptide G4 with the catalytic site of CD13. A Competitive inhibition of rCD13 enzymatic activity by peptide G4. Steady-state kinetic analysis of rCD13 in the presence of different concentrations of l -alanine p-nitroanilide hydrochloride substrate and G4, as indicated, in 60 mM potassium phosphate buffer, pH 7.4, at 37 °C, is shown. The initial velocities ( Vo ) were calculated from the slopes of the first 5 min of reaction. Two independent experiments, each in duplicate, were performed. The results of one representative experiment are shown in the left panel (mean ± SEM). The indicated enzyme inhibitory constant ( K i = 21 ± 7 nM , mean ± SEM of two independent experiments) was calculated using the competitive enzyme inhibition model of GraphPad Prism Software. The Lineweaver–Burk plot of the data (right panel) shows that G4 changed the apparent Km of the enzyme but not the Vmax, which is consistent with a competitive mechanism of inhibition. B Representative docking poses of G4 in complex with the closed (left) and intermediate open forms of human CD13 (center). For comparison, the crystallographic structure of CNGRCG in complex with the open form of porcine CD13 (pdb code: 4OU3) is shown on the right. CD13 and peptides are represented in cartoon and CPK, respectively. This model suggests that G4 fits into the catalytic pocket of the human CD13. C The best docking pose, in terms of ΔG MMGBSA , of G4 in complex with the closed and intermediate open form of human CD13 is shown on the left and on the right panels, respectively. CD13 residues involved in electrostatic interactions engaged by Val-1 are highlighted and labeled on the upper panels. The corresponding ligand interaction diagrams are shown in the lower panels. In the diagrams, protein pockets within 4 Å from G4 are displayed as colored spheres, where red, green, purple, and blue indicate acidic, hydrophobic, basic, and polar residues, respectively. H-bonds and salt bridges are shown as lines and arrows. G4 atoms exposed to solvent are highlighted with gray spheres

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: Inhibition, Activity Assay, Enzyme Inhibition Assay, Software, Comparison, Labeling, Solvent

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: G4 recognizes soluble forms of CD13, with higher affinity and selectivity than bestatin, and the membrane forms expressed on the surface of HUVEC cells. A Inhibitory effects of G4 and bestatin on the enzymatic activities of different aminopeptidases (rCD13, APA, and APB). Various amounts of G4 or bestatin were incubated with rCD13 (0.2 µg/mL in 60 mM Tris–HCl pH 7.4), APA (0.2 µg/mL in 60 mM Tris–HCl pH 7.4, 50 mM calcium chloride, 200 mM sodium chloride) or APB (0.2 µg/mL in 60 mM Tris–HCl pH 7.4, 100 mM potassium chloride) and the corresponding substrates at 0.1 mM ( l -alanine-7-amido-4-methylcoumarin for rCD13, l -glutamic acid γ-(7-amido-4-methylcoumarin) for APA, and l -arginine-7-amido-4-methylcoumarin for APB). The mixtures were incubated for 30 min at 37 °C, and the cleavage of each substrate was monitored by measuring the fluorescence of methylcoumarin ( λ ex 341 nm; λ em 441 nm) using an Infinite 200 PRO plate reader (Tecan). Three independent experiments were performed for each enzyme. The results of one representative experiment for each enzyme are shown (mean ± SEM, duplicates). B Inhibitory effects of G4 and bestatin on soluble CD13 immunocaptured from human serum (sCD13) using microtiter plates coated with anti-CD13 mAb WM15. The microtiter plate was filled with a solution of mAb WM15 (5 μg/ml in DPBS) and incubated overnight at 4 °C. The plate was then washed with DPBS, blocked with 3% bovine serum albumin (BSA) in DPBS, and filled with human serum containing soluble CD13 (diluted 1:2 with DPBS containing 2% BSA). After washing with DPBS, mixtures of G4 or bestatin (0–100 µM in 60 mM Tris–HCl, pH 7.4) and l -alanine p-nitroanilide hydrochloride substrate (4.5 mM in 60 mM Tris–HCl, pH 7.4) were added and left to incubate at 37 °C for 2 h. Substrate cleavage by sCD13 dissociated from mAb WM15 was then monitored by measuring the absorbance at 405 nm in each well using a plate reader (Bio-Rad). The results of one experiment performed in triplicate are shown (mean ± SEM). C Effect of G4 on the binding of the anti-CD13 mAb WM15 to HUVEC. HUVEC were seeded at a density of 2 × 10 5 cells in 100 µL DPBS supplemented with 5% normal human serum (NHS) and incubated with a mixture of G4 peptide (0, 0.1, 1, and 10 µM) and mAb WM15 (0 or 0.13 nM) as indicated. As a negative control, we used the 11-mer peptide CgA 429–439 , which is a human chromogranin A fragment. Cells were incubated for 60 min at 4 °C, washed, and resuspended in 100 µL of DPBS containing 5% NHS and 1 µg/mL FITC-labeled goat anti-mouse IgG secondary antibody. After incubation for 60 min at 4 °C, the cells were washed and analyzed by flow cytometry. Data are expressed as the percentage of counts vs. fluorescence intensity units

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: Membrane, Incubation, Fluorescence, Binding Assay, Negative Control, Labeling, Flow Cytometry

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: Effect of G4-TNF on tumor growth and body weight of mice bearing WEHI-164 fibrosarcomas. A Mice bearing subcutaneous WEHI-164 tumors (5–6 mice/groups) were treated 5 days after tumor implantation with a single intraperitoneal injection of G4-TNF (0, 4, 20, or 100 pg). Tumor volume and body weight after treatment were monitored daily. B Effect of mAb R3-63 (anti-murine CD13 antibody, 25 µg) and CNGRC peptide (100 µg) on the antitumor activity of G4-TNF (100 pg) (5–6 mice/group). The arrows indicate the time of treatment. * P < 0.05; ** P < 0.01, by Mann–Whitney analysis of the area under the curve for each tumor, with the GraphPad Prism software (mean ± SEM)

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: Tumor Implantation, Injection, Activity Assay, MANN-WHITNEY, Software

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: The active site of membrane CD13 expressed by HUVEC, but not that of soluble CD13 or membrane CD13 expressed by HL60 cells, is accessible to G4-TNF. A Competitive effects of G4 and G4-TNF on the binding of mAb WM15 (anti-CD13) to sCD13 or rCD13 showing the lack of binding of G4-TNF to natural and recombinant soluble CD13 (sCD13 and rCD13). G4, scrambled G4 (GCRSNCYRVG), and TNF were used as positive (G4) and negative (scrambled G4, TNF) controls. The sCD13 competition assay was performed as follows: microplates were coated with mAb WM15 (5 μg/mL) in DPBS and incubated overnight at 4 °C. Plates were subsequently blocked with 3% BSA in DPBS and filled with a mixture of human serum containing sCD13 (diluted 1:2) and the indicated competitors at different concentrations in DPBS, 0.1% BSA, and 0.05% Tween-20. The mixtures were incubated for 2 h at room temperature and washed with DPBS. To detect bound sCD13, we added an anti-CD13 rabbit polyclonal antibody (1:1000, 1 h at room temperature), washed with DPBS, and added peroxidase-labeled goat anti-rabbit secondary antibody (1:2000, 1 h). Bound peroxidase was detected using o-phenylendiamine chromogenic substrate. The absorbance of o-phenylenediamine was measured at 490 nm using an ELISA plate reader (Bio-Rad). Cumulative results of two independent experiments, each performed in duplicate, are shown (mean ± SEM). The rCD13 competition assay was performed as described in Supplementary Fig. S2 (one experiment in duplicate, mean ± SEM). B Inhibitory effects of G4 or G4-TNF on the sCD13 or rCD13 enzymatic activity (one experiment performed in duplicates; mean ± SEM). C Competition of mAb WM15 binding to HUVEC or HL-60 cells with G4 and G4-TNF. Scrambled G4 and TNF were used as negative controls. Cumulative results of three (HUVEC cells) or two (HL-60 cells) independent experiments, each performed in duplicate or triplicate (mean ± SEM). D Competitive binding of mAb WM15 (anti-CD13) to HUVEC and HL-60 cells with G4 and G4-Avidin complex prepared by mixing a G4-biotin conjugate with avidin (see “Methods). Avidin was also used as control. The experiment was performed in duplicate (mean ± SEM). E Inhibitory effects of G4 and G4-Avidin on rCD13 enzymatic activity. The assay was performed as described in Supplementary Fig. S2. The results of one experiment, performed in duplicate, are shown (mean ± SEM)

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: Membrane, Binding Assay, Recombinant, Competitive Binding Assay, Incubation, Labeling, Enzyme-linked Immunosorbent Assay, Activity Assay, Avidin-Biotin Assay

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: Quantification of CD13 expressed by HL-60 and HUVEC by western blotting and FACS analyses, and determination of their enzymatic activity. A Western blotting (WB) analysis of CD13 expression in HL-60 cells and HUVEC. For both cell lines, 2 µg of cell lysates were loaded in each well ( n = 4–5 replicates, as indicated). After blotting, the membrane was cut and analyzed for CD13 and actin expression as described in Material and methods. The results of one representative experiment, out of three independent experiments performed, are shown (bars, ratio of CD13/actin band intensity, mean ± SEM). Recombinant human CD13 (rCD13) (20 ng) was loaded as positive control. MW, molecular weight markers. B FACS analysis of CD13 expression in HUVEC and HL-60 cells. The cells were incubated with mAb WM15 (10 μg/mL) and stained with a FITC-labeled goat anti-mouse IgG (H + L) secondary antibody. Data were expressed as fluorescence intensity and normalized to the number of cells. The cumulative results of three independent experiments are shown (each performed in duplicate; mean ± SEM). C Enzyme activity assay of CD13 expressed in HUVEC and HL-60 cells. Cells were seeded into 96-well microtiter plates (3 × 10 5 cells/well) and incubated with l -alanine p-nitroanilide hydrochloride substrate (0.5 mM in DPBS) for 30 min at 37 °C. Absorbance at 405 nm was measured using a plate reader. The enzyme units in each sample were calculated using rCD13 as a standard and were normalized to the number of cells. Cumulative results of three independent experiments, each performed in duplicate, are shown (mean ± SEM). D Inhibition of the enzymatic activity of CD13 expressed in HUVEC and HL-60 cells by G4 and G4-TNF. Cells were seeded into 96-well microtiter plates (3 × 10 5 /well) and incubated with the l -alanine p-nitroanilide hydrochloride substrate and different amounts of G4-TNF, G4, TNF, or scrambled G4 peptide (30 min at 37 °C). The absorbance was measured at 405 nm using a plate reader. The results of one experiment performed in duplicate are shown (mean ± SEM)

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: Western Blot, Activity Assay, Expressing, Membrane, Recombinant, Positive Control, Molecular Weight, Incubation, Staining, Labeling, Fluorescence, Enzyme Activity Assay, Inhibition

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: Representative model of the interaction of G4 and G4-TNF with different human CD13 forms. Docking pose of G4 bound to CD13 in a closed and intermediate open form (left and center); model of G4-TNF in complex with the intermediate open form of CD13 (right). G4 and CD13 are represented by the pink and white surfaces, respectively. G4-TNF is shown in cyan

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: