mouse anti-human bid  (Proteintech)

Journal: International Journal of Biological Sciences

Article Title: Ganglioside GA2-mediated caspase-11 activation drives macrophage pyroptosis aggravating intimal hyperplasia after arterial injury

doi: 10.7150/ijbs.97106

Figure Lengend Snippet: GA2 mediates Cytochrome C-Casp9-Casp3 pathway activation through Cl-Casp4/11 binding to and cleaving BID. (A) Representative immunoblots of Casp8, Cl-Casp8, Casp9, Cl-Casp9, Casp3 and Cl-Casp3 in PMs and analysis results (n = 5). (B) Representative immunoblots of cytochrome C (Cyt C) in PM cytosol (Cyto) and mitochondrion (Mito) and analysis results (n = 5). (C) Representative immunoblots of BID, tBID (truncated BID), BIM and PUMA in PMs and analysis results (n = 5). (D-E) Coimmunoprecipitation (Co-IP) of activated Casp4 binds to BID in HEK293T cells (n = 3). HEK293T cells were transfected with the indicated plasmids for 24 h and the lysates incubated with anti-Flag-magnetic-beads, then immunoblotted with antibodies against Flag or HA. (F) Representative immunoblots of BID, tBID, Casp3, and Cl-Casp3 in PMs and analysis results (n = 6). (G) Representative immunoblots of BID, tBID, Casp3, and Cl-Casp3 in RAW264.7 cells and analysis results (n = 5). (H) Representative immunofluorescence staining sections from mice after 7 d of the injury for BID (light gray), F4/80 (green), and Casp11 (red) (n = 7). Nuclei were stained with DAPI (blue). Scale bars: 100 μm. A , B , C , F (tBID) and G (tBID) were tested using a One-way ANOVA test; F (Cl-Casp3) and G (Cl-Casp3) were tested using a Welch's ANOVA test.

Article Snippet: The following primary antibodies were used for western blot: MLKL (1:1000, 37333S, CST), Phospho-MLKL (1:1000, 37333S, CST), RIPK3 (1:1000, 95702S, CST), Phospho-RIPK3 (1:1000, 91702S, CST), GSDMD (1:500, sc-393581, Santa Cruz), GSDME (1:1000, ab215191, Abcam), caspase-11 (1:1000, ab180673, Abcam), caspase-1 (1:1000, ab179515, Abcam), caspase-4 (1:1000, M029-3, MBL), caspase-8 (1:1000, 9746T, CST), caspase-9 (1:1000, 9508T, CST), caspase-3 (1:1000, 9662S, CST), cytochrome C (1:1000, 11940S, CST), BID (1:1000, AF860, R&D Systems), BIM (1:500, sc-374358, Santa Cruz), PUMA (1:500, sc-374223, Santa Cruz), COX IV (1:5000, 11242-1-AP, Proteintech), β-actin (1:5000, BS6007M, Bioworld).

Techniques: Activation Assay, Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Transfection, Incubation, Magnetic Beads, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: ( A ) Quantification of internalized B19V. Lentivirus-transduced UT7/Epo-S1 cells (S1 shAXL or S1 shScr ) were incubated with B19V at an MOI of 1000 at 37°C for 1.5 hours. After treatment with trypsin and benzonase, the cells were washed with PBS three times, and viral DNA was extracted for quantification by qPCR. ( B to D ) Quantification of B19V replication and egress. S1 shAXL or S1 shScr cells were infected with B19V at an MOI of 1000. At 2 dpi, the cells were collected for quantification of B19V replication by relative viral DNA levels versus mitochondrial DNA (B), relative VP2 mRNA versus β-actin mRNA (C), or virus egress (D). The values (mean ± SD) obtained from S1 shAXL are normalized to those from the S1 shScr cells, which are arbitrarily set up to 100% [(D), S1 shScr ]. All the experiments were performed in triplicate, and the data were analyzed by Student’s t test (** P < 0.01 and **** P < 0.0001).

Article Snippet: The following primary antibodies were purchased: mouse monoclonal antibody of anti-B19V capsid (#MAB8292, clone 521-5D) and mouse anti–β-actin (#A5441) from MilliporeSigma; mouse monoclonal antibody against B19V conformational epitope in VP2 (MAB 860-55D) from MIKROGEN (Neuried, Germany); goat anti-AXL antibody (#AF154) from R&D Systems Minneapolis, MN); rabbit anti-AXL (#8661S, clone C89E7), anti-Ku80 (#2180), and anti-integrin α5 (#98204) from Cell Signaling Technology; and mouse anti-Flag (#200-301-B13) and rabbit anti-BrdU (#600-401-C29) from Rockland, Limerick, PA. Anti-B19V VP1u antibody was in-house prepared.

Techniques: Incubation, Infection, Virus

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: Lentivirus-transduced CD36 + EPCs were incubated with B19V at an MOI of 1000 at 37°C for 1.5 hours. ( A ) Quantification of internalized B19V. Cells were treated with trypsin and benzonase, followed by washing with PBS. The internalized B19V was quantified by qPCR. ( B and C ) Quantification of B19V replication. At 2 dpi, the indicated cells were collected and treated with benzonase, followed by quantification of B19V DNA using qPCR (B) and VP2 mRNA using RT-qPCR (C). ( D ) Immunofluorescence assay. At 2 dpi, cells were collected and resuspended in DMEM and cytospun onto slides, followed by fixation, permeabilization, and sequentially staining with anti-B19V capsid (clone 521-5D) and an FITC-conjugated secondary antibody (green). Confocal images were taken at a magnification of ×20. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E and F ) Flow cytometry. At 2 dpi, infected EPCs were fixed, permeabilized, and stained with anti-B19V capsid (#clone 521-5D) and an FITC-conjugated secondary antibody, sequentially, followed by flow cytometry (E). All the experiments were performed in triplicate (F), and the data shown were analyzed by Student’s t test (*** P < 0.001 and **** P < 0.0001).

Article Snippet: The following primary antibodies were purchased: mouse monoclonal antibody of anti-B19V capsid (#MAB8292, clone 521-5D) and mouse anti–β-actin (#A5441) from MilliporeSigma; mouse monoclonal antibody against B19V conformational epitope in VP2 (MAB 860-55D) from MIKROGEN (Neuried, Germany); goat anti-AXL antibody (#AF154) from R&D Systems Minneapolis, MN); rabbit anti-AXL (#8661S, clone C89E7), anti-Ku80 (#2180), and anti-integrin α5 (#98204) from Cell Signaling Technology; and mouse anti-Flag (#200-301-B13) and rabbit anti-BrdU (#600-401-C29) from Rockland, Limerick, PA. Anti-B19V VP1u antibody was in-house prepared.

Techniques: Incubation, Quantitative RT-PCR, Immunofluorescence, Staining, Flow Cytometry, Infection

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: ( A and D ) Quantification of B19V DNA replication. (A) CD36 + EPCs were incubated with an anti-AXL or an IgG isotype control (at indicated concentrations) at 4°C before B19V infection (MOI = 1000). (D) CD36 + EPCs were incubated with soluble rAXL EC or MBP (0.1 to 10 μg/ml) during B19V infection. At 2 dpi, the cells were collected and quantified for replicated B19V genome and mitochondrial DNA using qPCR. The number of replicated B19V genomes of each group is standardized with the mitochondrial DNA numbers. Data shown are relative to the mock-treated cell group, which is arbitrarily set up as 100%. ( B and C ) Immunofluorescence assay and flow cytometry. CD36 + EPCs were incubated with anti-AXL or IgG isotype control (10 μg/ml) at 4°C before B19V infection. At 2 dpi, the cells were collected and stained with an anti-capsid antibody (clone 521-5D), followed by immunofluorescence assay (B) and flow cytometry (C), respectively. Scale bar, 50 μm.

Article Snippet: The following primary antibodies were purchased: mouse monoclonal antibody of anti-B19V capsid (#MAB8292, clone 521-5D) and mouse anti–β-actin (#A5441) from MilliporeSigma; mouse monoclonal antibody against B19V conformational epitope in VP2 (MAB 860-55D) from MIKROGEN (Neuried, Germany); goat anti-AXL antibody (#AF154) from R&D Systems Minneapolis, MN); rabbit anti-AXL (#8661S, clone C89E7), anti-Ku80 (#2180), and anti-integrin α5 (#98204) from Cell Signaling Technology; and mouse anti-Flag (#200-301-B13) and rabbit anti-BrdU (#600-401-C29) from Rockland, Limerick, PA. Anti-B19V VP1u antibody was in-house prepared.

Techniques: Incubation, Control, Infection, Immunofluorescence, Flow Cytometry, Staining

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: ( A ) Immunofluorescent assay for colocalization of AXL with B19V virions. CD36 + EPCs cells were infected with B19V at an MOI of ~5000 vgc per cell for 1.5 hours at 4°C. The cells were then washed with ice-cold PBS, then cytospun onto slides, fixed, permeabilized, and stained with an anti-B19V capsid antibody (clone MAB860-55D). Confocal images were taken under a confocal microscope. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. ( B to D ) Kinetics of the in vitro interaction between AXL and VP1u using Ni-NTA biosensors. The binding kinetics depicts the associations and dissociations of 4 μM rAXL EC with GST-VP1u at the indicated concentrations (B). Binding kinetics of 4 μM AXL His with 4 and 8 μM GST was used as a control (C). The binding parameters of AXL and GST-VP1u are shown (D). Equilibrium dissociation constant K D value represents the ratio of dissociation [ K d (1/s)] and association [ K a (1/Ms)] computed from the real-time binding curves of rAXL EC to GST-VP1u. The values are shown with means ± SDs based on at least three repeated experiments.

Article Snippet: The following primary antibodies were purchased: mouse monoclonal antibody of anti-B19V capsid (#MAB8292, clone 521-5D) and mouse anti–β-actin (#A5441) from MilliporeSigma; mouse monoclonal antibody against B19V conformational epitope in VP2 (MAB 860-55D) from MIKROGEN (Neuried, Germany); goat anti-AXL antibody (#AF154) from R&D Systems Minneapolis, MN); rabbit anti-AXL (#8661S, clone C89E7), anti-Ku80 (#2180), and anti-integrin α5 (#98204) from Cell Signaling Technology; and mouse anti-Flag (#200-301-B13) and rabbit anti-BrdU (#600-401-C29) from Rockland, Limerick, PA. Anti-B19V VP1u antibody was in-house prepared.

Techniques: Infection, Staining, Microscopy, In Vitro, Binding Assay, Control

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: ( A ) Cell surface expression of AXL. K562 cells were transduced with lentivirus-expressing AXL protein to express AXL on the cell surface (K562 AXL ). Blank lentivirus vector serves as control (K562 lentictrl ). Flow cytometry was used to analyze cell surface expression of AXL. ( B and C ) Overexpression of AXL on K562 cells increases B19V entry and infection. K562, K562 AXL , and K562 lentictrl cells were infected with B19V at an MOI of ~2000 at 37°C for 1.5 hours. (B) Internalized B19V virions were quantified by qPCR. (C) At 2 dpi, total DNA was extracted and analyzed for the replicated viral DNA and mitochondrial DNA by qPCR. The numbers shown are mitochondrial DNA–standardized B19V genome levels of the infected K562 AXL and K562 lentictrl cells related to that of the control K562 cells.

Article Snippet: The following primary antibodies were purchased: mouse monoclonal antibody of anti-B19V capsid (#MAB8292, clone 521-5D) and mouse anti–β-actin (#A5441) from MilliporeSigma; mouse monoclonal antibody against B19V conformational epitope in VP2 (MAB 860-55D) from MIKROGEN (Neuried, Germany); goat anti-AXL antibody (#AF154) from R&D Systems Minneapolis, MN); rabbit anti-AXL (#8661S, clone C89E7), anti-Ku80 (#2180), and anti-integrin α5 (#98204) from Cell Signaling Technology; and mouse anti-Flag (#200-301-B13) and rabbit anti-BrdU (#600-401-C29) from Rockland, Limerick, PA. Anti-B19V VP1u antibody was in-house prepared.

Techniques: Expressing, Transduction, Plasmid Preparation, Control, Flow Cytometry, Over Expression, Infection

Journal: Science Advances

Article Title: Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors

doi: 10.1126/sciadv.ade0869

Figure Lengend Snippet: ( A ) Immunofluorescent assay for colocalization of AXL with B19V virions. CD36 + EPCs cells were infected with B19V at an MOI of ~5000 vgc per cell for 1.5 hours at 4°C. The cells were then washed with ice-cold PBS, then cytospun onto slides, fixed, permeabilized, and stained with an anti-B19V capsid antibody (clone MAB860-55D). Confocal images were taken under a confocal microscope. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. ( B to D ) Kinetics of the in vitro interaction between AXL and VP1u using Ni-NTA biosensors. The binding kinetics depicts the associations and dissociations of 4 μM rAXL EC with GST-VP1u at the indicated concentrations (B). Binding kinetics of 4 μM AXL His with 4 and 8 μM GST was used as a control (C). The binding parameters of AXL and GST-VP1u are shown (D). Equilibrium dissociation constant K D value represents the ratio of dissociation [ K d (1/s)] and association [ K a (1/Ms)] computed from the real-time binding curves of rAXL EC to GST-VP1u. The values are shown with means ± SDs based on at least three repeated experiments.

Article Snippet: The fixed cells were then incubated with an anti-B19V capsid antibody [#MAB8292/clone 521-5D ( ) or #MAb860-55D ( , )] to detect intact B19V virions or costained with anti-AXL antibody (#AF154, R&D Systems).

Techniques: Infection, Staining, Microscopy, In Vitro, Binding Assay, Control