anti gapdh primary ab  (BioLegend)

 
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    Name:
    Anti GAPDH
    Description:
    Anti GAPDH 1D4 Isotype Mouse IgM κ Reactivity Mammalian including Human Cow Pig Mouse Rat Avian Apps WB IF ICC Size 200 μl
    Catalog Number:
    919501
    Price:
    266
    Applications:
    WB, IF, ICC
    Conjugate:
    TCS
    Immunogen:
    This antibody was raised against extensively purified pig GAPDH.
    Size:
    200 μl
    Preparation:
    Tissue Culture Supernatant
    Source:
    Mouse
    Quantity:
    1
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    Structured Review

    BioLegend anti gapdh primary ab
    Anti GAPDH
    Anti GAPDH 1D4 Isotype Mouse IgM κ Reactivity Mammalian including Human Cow Pig Mouse Rat Avian Apps WB IF ICC Size 200 μl
    https://www.bioz.com/result/anti gapdh primary ab/product/BioLegend
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti gapdh primary ab - by Bioz Stars, 2020-09
    99/100 stars

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    Related Articles

    Staining:

    Article Title: Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells
    Article Snippet: .. Proteins were stained using a mouse anti-FLAG (Sigma Aldrich, #F1804), rabbit anti-HA (Cell Signaling, #3724), rabbit anti-GFP/BFP (Abcam, #ab290) and rat anti-GAPDH (Biolegend, #607902) antibodies. .. To confirm removal of nucleic acids, 20 μl of lysates were diluted 1:5 with Roti-Load DNA (with glycerol) (Roth, #X904.1) and run on a 1% agarose gel (VWR-Peqlab, #9012-36-6) supplemented with 1 drop of ethidium bromide (ITW Reagents, #1239-45-8) for 25 minutes.

    Flow Cytometry:

    Article Title: LFA-1-targeting Leukotoxin (LtxA; Leukothera®) causes lymphoma tumor regression in a humanized mouse model and requires caspase-8 and Fas to kill malignant lymphocytes*
    Article Snippet: .. Anti-human cleaved caspase-8 (clone 11G10), anti-human caspase-8 (clone IC12) (both from Cell Signaling), anti-human caspase-9 (clone LAP6, R & D Systems), and anti-GAPDH (cloneFF26A/F9 from BioLegend) were the primary antibodies used for immunoblot analysis or flow cytometry. .. The secondary antibody (1:200) used to detect cleaved caspase-8 was Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen).

    SDS Page:

    Article Title: Regulation of CCR7-dependent cell migration through CCR7 homodimer formation
    Article Snippet: .. The samples were separated by SDS-PAGE, and transferred onto PVDF membranes for immunoblotting with an anti-phospho-Akt, phospho-Erk1/2, total-Akt, total-Erk1/2 antibody (Cell Signaling Inc., MA, USA) or anti-GAPDH antibody (Biolegend). .. Bound antibodies were detected using the ECL system (GE Healthcare Life Sciences).

    Incubation:

    Article Title: Foreign RNA Induces the Degradation of Mitochondrial Antiviral Signaling Protein (MAVS): The Role of Intracellular Antiviral Factors
    Article Snippet: .. The membranes were incubated overnight at 4°C with one of the following primary antibodies: mouse anti-RIG-I, mouse anti-Cardif (MAVS) (Enzo Life Sciences, Miami, FL), rabbit anti-IRF3 (IBL, Japan), mouse anti-GAPDH (BioLegend, San Diego, CA) or rabbit anti-β-actin (Sigma-Aldrich). .. After five washes with TBST, the membranes were further incubated for 1 h at room temperature with bovine anti-rabbit (Santa Cruz Biotechnology, Santa Cruz, CA) or Zymax anti-mouse IgG antibody (Invitrogen) coupled with HRP at a 1∶10,000 dilution in blocking buffer.

    Cytometry:

    Article Title: LFA-1-targeting Leukotoxin (LtxA; Leukothera®) causes lymphoma tumor regression in a humanized mouse model and requires caspase-8 and Fas to kill malignant lymphocytes*
    Article Snippet: .. Anti-human cleaved caspase-8 (clone 11G10), anti-human caspase-8 (clone IC12) (both from Cell Signaling), anti-human caspase-9 (clone LAP6, R & D Systems), and anti-GAPDH (cloneFF26A/F9 from BioLegend) were the primary antibodies used for immunoblot analysis or flow cytometry. .. The secondary antibody (1:200) used to detect cleaved caspase-8 was Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen).

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  • 91
    BioLegend purified anti gapdh
    TMPRSS2, TMPRSS4 but not ST14 mediate SARS-CoV-2 entry (A) Bulk RNA-sequencing results of intestine-specific serine protease expression in HEK293, Huh7.5, H1-Hela, HT-29 cells and human ileum enteroids. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left panel), or transfected with indicated plasmid combination for 24 hours (right panel), and infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of <t>GAPDH.</t> (C) HEK293 cells stably expressing human ACE2 were transfected with C-terminally tagged SARS-CoV-2 S and TMPRSS2 or TMPRSS4 or 48 hours. The levels of S and GAPDH were measured by western blot. The intensity of bands were quantified by ImageJ and shown as percentage of the top band in lane 1. (D) HEK293 cells stably expressing human ACE2 were transfected with TMPRSS2 or TMPRSS4 for 24 hours, incubated with 5.8×10 5 PFUs of SARS-CoV-2 chimera virus on ice for 1 hour, washed with cold PBS for 3 times, and shifted to 37 °C for another hour. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Wild-type or human ACE2 expressing HEK293 cells were transfected with SARS-CoV-2 S and <t>GFP,</t> with or without TMPRSS2 or TMPRSS4 or 24 hours. The red arrows highlight the formation of large syncytia.
    Purified Anti Gapdh, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purified anti gapdh/product/BioLegend
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    purified anti gapdh - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    99
    BioLegend anti gapdh biolegend primary antibodies
    TMPRSS2, TMPRSS4 but not ST14 mediate SARS-CoV-2 entry (A) Bulk RNA-sequencing results of intestine-specific serine protease expression in HEK293, Huh7.5, H1-Hela, HT-29 cells and human ileum enteroids. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left panel), or transfected with indicated plasmid combination for 24 hours (right panel), and infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of <t>GAPDH.</t> (C) HEK293 cells stably expressing human ACE2 were transfected with C-terminally tagged SARS-CoV-2 S and TMPRSS2 or TMPRSS4 or 48 hours. The levels of S and GAPDH were measured by western blot. The intensity of bands were quantified by ImageJ and shown as percentage of the top band in lane 1. (D) HEK293 cells stably expressing human ACE2 were transfected with TMPRSS2 or TMPRSS4 for 24 hours, incubated with 5.8×10 5 PFUs of SARS-CoV-2 chimera virus on ice for 1 hour, washed with cold PBS for 3 times, and shifted to 37 °C for another hour. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Wild-type or human ACE2 expressing HEK293 cells were transfected with SARS-CoV-2 S and <t>GFP,</t> with or without TMPRSS2 or TMPRSS4 or 24 hours. The red arrows highlight the formation of large syncytia.
    Anti Gapdh Biolegend Primary Antibodies, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gapdh biolegend primary antibodies/product/BioLegend
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    anti gapdh biolegend primary antibodies - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    TMPRSS2, TMPRSS4 but not ST14 mediate SARS-CoV-2 entry (A) Bulk RNA-sequencing results of intestine-specific serine protease expression in HEK293, Huh7.5, H1-Hela, HT-29 cells and human ileum enteroids. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left panel), or transfected with indicated plasmid combination for 24 hours (right panel), and infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (C) HEK293 cells stably expressing human ACE2 were transfected with C-terminally tagged SARS-CoV-2 S and TMPRSS2 or TMPRSS4 or 48 hours. The levels of S and GAPDH were measured by western blot. The intensity of bands were quantified by ImageJ and shown as percentage of the top band in lane 1. (D) HEK293 cells stably expressing human ACE2 were transfected with TMPRSS2 or TMPRSS4 for 24 hours, incubated with 5.8×10 5 PFUs of SARS-CoV-2 chimera virus on ice for 1 hour, washed with cold PBS for 3 times, and shifted to 37 °C for another hour. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Wild-type or human ACE2 expressing HEK293 cells were transfected with SARS-CoV-2 S and GFP, with or without TMPRSS2 or TMPRSS4 or 24 hours. The red arrows highlight the formation of large syncytia.

    Journal: bioRxiv

    Article Title: TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

    doi: 10.1101/2020.04.21.054015

    Figure Lengend Snippet: TMPRSS2, TMPRSS4 but not ST14 mediate SARS-CoV-2 entry (A) Bulk RNA-sequencing results of intestine-specific serine protease expression in HEK293, Huh7.5, H1-Hela, HT-29 cells and human ileum enteroids. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left panel), or transfected with indicated plasmid combination for 24 hours (right panel), and infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (C) HEK293 cells stably expressing human ACE2 were transfected with C-terminally tagged SARS-CoV-2 S and TMPRSS2 or TMPRSS4 or 48 hours. The levels of S and GAPDH were measured by western blot. The intensity of bands were quantified by ImageJ and shown as percentage of the top band in lane 1. (D) HEK293 cells stably expressing human ACE2 were transfected with TMPRSS2 or TMPRSS4 for 24 hours, incubated with 5.8×10 5 PFUs of SARS-CoV-2 chimera virus on ice for 1 hour, washed with cold PBS for 3 times, and shifted to 37 °C for another hour. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Wild-type or human ACE2 expressing HEK293 cells were transfected with SARS-CoV-2 S and GFP, with or without TMPRSS2 or TMPRSS4 or 24 hours. The red arrows highlight the formation of large syncytia.

    Article Snippet: Primary antibodies used in this study included: GAPDH (631402, Biolegend); GFP (2555S, Cell Signaling); SARS-CoV-2-S (S1) (PA5-81795, Thermo Fisher); TMPRSS2 (sc-515727, Santa Cruz); TMPRSS4 (sc-376415, Santa Cruz); and V5 (13202S, Cell Signaling).

    Techniques: RNA Sequencing Assay, Expressing, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Stable Transfection, Western Blot, Incubation

    TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection in enteroids (A) Schematic diagram of SARS-CoV-2 infection of human mature enterocytes (left panel). An HEK293 stable cell line expressing ACE2 and TMPRSS4 were transfected with GFP and an HEK293 stable cell line expressing TMPRSS2 were transfected with SARS-CoV-2 S and TdTomato for 24 hours. These two cell lines were then mixed at 1:1 ratio and cultured for another 24 hours. Note the formation of cell-cell fusion (yellow), highlighted by black arrows. (B) Human duodenum enteroids in 3D Matrigel were transduced with lentiviruses encoding Cas9 and sgRNA against TMPRSS2 or TMPRSS4 (oligonucleotide information in Table S1). Gene knockout enteroids were seeded into monolayers and infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (C) Human duodenum enteroids seeded into collagen-coated 96-well plates were differentiated for 3 days, pre-treated with 50 μg/ml of soybean trypsin inhibitor (SBTI), 10 μM of camostat mesylate, or 10 μM of E-64d for 30 minutes, and infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH.

    Journal: bioRxiv

    Article Title: TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

    doi: 10.1101/2020.04.21.054015

    Figure Lengend Snippet: TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection in enteroids (A) Schematic diagram of SARS-CoV-2 infection of human mature enterocytes (left panel). An HEK293 stable cell line expressing ACE2 and TMPRSS4 were transfected with GFP and an HEK293 stable cell line expressing TMPRSS2 were transfected with SARS-CoV-2 S and TdTomato for 24 hours. These two cell lines were then mixed at 1:1 ratio and cultured for another 24 hours. Note the formation of cell-cell fusion (yellow), highlighted by black arrows. (B) Human duodenum enteroids in 3D Matrigel were transduced with lentiviruses encoding Cas9 and sgRNA against TMPRSS2 or TMPRSS4 (oligonucleotide information in Table S1). Gene knockout enteroids were seeded into monolayers and infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (C) Human duodenum enteroids seeded into collagen-coated 96-well plates were differentiated for 3 days, pre-treated with 50 μg/ml of soybean trypsin inhibitor (SBTI), 10 μM of camostat mesylate, or 10 μM of E-64d for 30 minutes, and infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH.

    Article Snippet: Primary antibodies used in this study included: GAPDH (631402, Biolegend); GFP (2555S, Cell Signaling); SARS-CoV-2-S (S1) (PA5-81795, Thermo Fisher); TMPRSS2 (sc-515727, Santa Cruz); TMPRSS4 (sc-376415, Santa Cruz); and V5 (13202S, Cell Signaling).

    Techniques: Infection, Stable Transfection, Expressing, Transfection, Cell Culture, Transduction, Gene Knockout, Quantitative RT-PCR

    ACE2 is highly expressed in the human intestine (A) ACE2 expression in different human tissues and organs from human protein atlas ( www.proteinatlas.org/ ). (B) Ace2 expression in different mouse tissues and cell types from BioGPS ( biogps.org ). (C) Bulk RNA-sequencing results of ACE2 and other CoV receptor expression in HEK293, Huh7.5, H1-Hela, HT-29 cells and human ileum enteroids. (D) ACE2 expression level in HEK293, HT-29 cells, duodenum enteroids and ileum enteroids were measured by RT-qPCR and normalized to that of GAPDH. (E) Human duodenum enteroids in 3D Matrigel were cultured in differentiation media for 3 days and stained for ACE2 (red), villin (green), and nucleus (DAPI, blue). Scale bar: 50 μm. (F) Differentiated human duodenum enteroids were allowed to flip inside out, infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. Enteroids were stained for virus (green), actin (phalloidin, grey), and nucleus (DAPI, blue). Scale bar: 32 μm.

    Journal: bioRxiv

    Article Title: TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

    doi: 10.1101/2020.04.21.054015

    Figure Lengend Snippet: ACE2 is highly expressed in the human intestine (A) ACE2 expression in different human tissues and organs from human protein atlas ( www.proteinatlas.org/ ). (B) Ace2 expression in different mouse tissues and cell types from BioGPS ( biogps.org ). (C) Bulk RNA-sequencing results of ACE2 and other CoV receptor expression in HEK293, Huh7.5, H1-Hela, HT-29 cells and human ileum enteroids. (D) ACE2 expression level in HEK293, HT-29 cells, duodenum enteroids and ileum enteroids were measured by RT-qPCR and normalized to that of GAPDH. (E) Human duodenum enteroids in 3D Matrigel were cultured in differentiation media for 3 days and stained for ACE2 (red), villin (green), and nucleus (DAPI, blue). Scale bar: 50 μm. (F) Differentiated human duodenum enteroids were allowed to flip inside out, infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. Enteroids were stained for virus (green), actin (phalloidin, grey), and nucleus (DAPI, blue). Scale bar: 32 μm.

    Article Snippet: Primary antibodies used in this study included: GAPDH (631402, Biolegend); GFP (2555S, Cell Signaling); SARS-CoV-2-S (S1) (PA5-81795, Thermo Fisher); TMPRSS2 (sc-515727, Santa Cruz); TMPRSS4 (sc-376415, Santa Cruz); and V5 (13202S, Cell Signaling).

    Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Cell Culture, Staining, Infection

    SARS-CoV-2 actively replicates in ACE2+ human mature enterocytes (A) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically or basolaterally infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (B) Supernatants in both apical and basal chambers were collected from (A) and were subjected to an TCID50 assay to measure the amount of infectious viruses. (C) Differentiated duodenum enteroids in monolayer were apically infected with 2.5×10 5 PFUs of infectious SARS-CoV-2 virus (MOI=0.5) for 8 hours. The expression of SARS-CoV-2 N was measured by RT-qPCR using a Taqman assay and normalized to that of GAPDH. (D) Same as (C) except that enteroids were fixed and stained for SARS-CoV-2 S (green), ACE2 (red), and nucleus (DAPI, blue). Scale bar: 32 μm. SARS-CoV-2 infected ACE2 positive cells are enlarged in the inset (yellow box). (E) SARS-CoV-2 infected duodenum monolayers were imaged along the z-stacks and sectioned for YZ planes (top panel) and reconstructed for 3D images (bottom panel).

    Journal: bioRxiv

    Article Title: TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

    doi: 10.1101/2020.04.21.054015

    Figure Lengend Snippet: SARS-CoV-2 actively replicates in ACE2+ human mature enterocytes (A) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically or basolaterally infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (B) Supernatants in both apical and basal chambers were collected from (A) and were subjected to an TCID50 assay to measure the amount of infectious viruses. (C) Differentiated duodenum enteroids in monolayer were apically infected with 2.5×10 5 PFUs of infectious SARS-CoV-2 virus (MOI=0.5) for 8 hours. The expression of SARS-CoV-2 N was measured by RT-qPCR using a Taqman assay and normalized to that of GAPDH. (D) Same as (C) except that enteroids were fixed and stained for SARS-CoV-2 S (green), ACE2 (red), and nucleus (DAPI, blue). Scale bar: 32 μm. SARS-CoV-2 infected ACE2 positive cells are enlarged in the inset (yellow box). (E) SARS-CoV-2 infected duodenum monolayers were imaged along the z-stacks and sectioned for YZ planes (top panel) and reconstructed for 3D images (bottom panel).

    Article Snippet: Primary antibodies used in this study included: GAPDH (631402, Biolegend); GFP (2555S, Cell Signaling); SARS-CoV-2-S (S1) (PA5-81795, Thermo Fisher); TMPRSS2 (sc-515727, Santa Cruz); TMPRSS4 (sc-376415, Santa Cruz); and V5 (13202S, Cell Signaling).

    Techniques: Cell Culture, Infection, Expressing, Quantitative RT-PCR, TCID50 Assay, TaqMan Assay, Staining

    SARS-CoV-2 S chimera virus infects human small intestinal enteroids (A) Mouse small intestinal cells were analyzed by single-cell RNA-sequencing and resolved into 20 clusters based on gene expression profiles (left panel). Transcript levels of Cd26, Epcam, Cd44, Cd45, and Ace2 were indicated for different intestinal cell subsets. Each dot represents a single cell. Note that Ace2 high cells are also positive for Cd26 and Epcam but negative for Cd44 and Cd45. (B) Human duodenum enteroids were cultured in the transwell monolayer system using maintenance (MAINT) or differentiation (DIFF) conditions for 3 days. Monolayers were stained for ACE2 (red) and actin (phalloidin, white). Scale bar: 32 μm. (C) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5×10 5 plaque forming units (PFUs) of SARS-CoV-2 chimera virus (MOI=0.3) for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (D) Human duodenum enteroids in 3D Matrigel were cultured in maintenance (MAINT) media or differentiation (DIFF) media for 3 days and infected with 2.2×10 5 PFUs of SARS-CoV-2 chimera virus for 18 hours. Enteroids were stained for virus (green), actin (phalloidin, white), and nucleus (DAPI, blue). Scale bar: 50 μm. (E) Same as (C) except that virus titers were measured using an TCID50 assay instead of viral RNA levels by QPCR. (F) Same as (D) except that human ileum enteroids were used instead. Scale bar: 80 μm. (G) Same as (D) except that human ileum enteroids were used instead. Scale bar: 80 μm.

    Journal: bioRxiv

    Article Title: TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

    doi: 10.1101/2020.04.21.054015

    Figure Lengend Snippet: SARS-CoV-2 S chimera virus infects human small intestinal enteroids (A) Mouse small intestinal cells were analyzed by single-cell RNA-sequencing and resolved into 20 clusters based on gene expression profiles (left panel). Transcript levels of Cd26, Epcam, Cd44, Cd45, and Ace2 were indicated for different intestinal cell subsets. Each dot represents a single cell. Note that Ace2 high cells are also positive for Cd26 and Epcam but negative for Cd44 and Cd45. (B) Human duodenum enteroids were cultured in the transwell monolayer system using maintenance (MAINT) or differentiation (DIFF) conditions for 3 days. Monolayers were stained for ACE2 (red) and actin (phalloidin, white). Scale bar: 32 μm. (C) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5×10 5 plaque forming units (PFUs) of SARS-CoV-2 chimera virus (MOI=0.3) for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (D) Human duodenum enteroids in 3D Matrigel were cultured in maintenance (MAINT) media or differentiation (DIFF) media for 3 days and infected with 2.2×10 5 PFUs of SARS-CoV-2 chimera virus for 18 hours. Enteroids were stained for virus (green), actin (phalloidin, white), and nucleus (DAPI, blue). Scale bar: 50 μm. (E) Same as (C) except that virus titers were measured using an TCID50 assay instead of viral RNA levels by QPCR. (F) Same as (D) except that human ileum enteroids were used instead. Scale bar: 80 μm. (G) Same as (D) except that human ileum enteroids were used instead. Scale bar: 80 μm.

    Article Snippet: Primary antibodies used in this study included: GAPDH (631402, Biolegend); GFP (2555S, Cell Signaling); SARS-CoV-2-S (S1) (PA5-81795, Thermo Fisher); TMPRSS2 (sc-515727, Santa Cruz); TMPRSS4 (sc-376415, Santa Cruz); and V5 (13202S, Cell Signaling).

    Techniques: RNA Sequencing Assay, Expressing, Cell Culture, Staining, Infection, Quantitative RT-PCR, TCID50 Assay, Real-time Polymerase Chain Reaction

    TMPRSS2 and TMPRSS4 enhance SARS-CoV-2 infectivity (A) Transcript levels of Ace2, Tmprss2, Tmprss4, and St14 were indicated for different intestinal cell subsets. Each dot represents a single cell. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left panel), or transfected with indicated plasmid combination for 24 hours (right panel). The levels of V5 and GAPDH were measured by western blot. (C) HEK293 cells were transfected with indicated plasmid combination for 24 hours (right panel), and infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The amount of infectious viruses was measured using an TCID50 assay. (D) Same as (C) except that virus-infected cells (GFP) were imaged. (E) Same as (C) except that the levels of V5, GFP, and GAPDH were measured by western blot.

    Journal: bioRxiv

    Article Title: TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

    doi: 10.1101/2020.04.21.054015

    Figure Lengend Snippet: TMPRSS2 and TMPRSS4 enhance SARS-CoV-2 infectivity (A) Transcript levels of Ace2, Tmprss2, Tmprss4, and St14 were indicated for different intestinal cell subsets. Each dot represents a single cell. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left panel), or transfected with indicated plasmid combination for 24 hours (right panel). The levels of V5 and GAPDH were measured by western blot. (C) HEK293 cells were transfected with indicated plasmid combination for 24 hours (right panel), and infected with 1.5×10 5 PFUs of SARS-CoV-2 chimera virus for 24 hours. The amount of infectious viruses was measured using an TCID50 assay. (D) Same as (C) except that virus-infected cells (GFP) were imaged. (E) Same as (C) except that the levels of V5, GFP, and GAPDH were measured by western blot.

    Article Snippet: Primary antibodies used in this study included: GAPDH (631402, Biolegend); GFP (2555S, Cell Signaling); SARS-CoV-2-S (S1) (PA5-81795, Thermo Fisher); TMPRSS2 (sc-515727, Santa Cruz); TMPRSS4 (sc-376415, Santa Cruz); and V5 (13202S, Cell Signaling).

    Techniques: Infection, Transfection, Plasmid Preparation, Western Blot, TCID50 Assay