Journal: Cell & Bioscience

Article Title: Development of a novel human CD147 knock-in NSG mouse model to test SARS-CoV-2 viral infection

doi: 10.1186/s13578-022-00822-6

Figure Lengend Snippet: Schematic representation of genotyping primers used to confirm hCD147KI expression. A combination of 4 primers was used to screen mice (internal primers hCD147A and hCD147B) and confirm proper integration into the mouse CD147 allele (flanking primers BSGC and BSGD)

Article Snippet: To stain mouse CD147 (mCD147), polyclonal donkey anti-goat IgG secondary (Jackson ImmunoResearch; 705–035-003 [HRP]; 1:500) was used against primary goat anti-mouse CD147 antibody (R&D Systems; BAF772; 1:100).

Techniques: Expressing

Journal: Cell & Bioscience

Article Title: Development of a novel human CD147 knock-in NSG mouse model to test SARS-CoV-2 viral infection

doi: 10.1186/s13578-022-00822-6

Figure Lengend Snippet: H&E and IHC of human CD147 in hCD147KI het -NSG mice. Human CD147 was stained (HIM6; 1:500) in the ( A ) lung, ( B ) liver, ( C ) intestine, ( D ) heart, ( E ) brain, ( F ) spleen, ( G ) kidney, ( H ) testis, and ( I ) trachea in WT-NSG (top) and hCD147KI het -NSG (bottom) mice. Images were taken using an Olympus Inverted Light Microscope. Scale bar represents 100 µm

Article Snippet: To stain mouse CD147 (mCD147), polyclonal donkey anti-goat IgG secondary (Jackson ImmunoResearch; 705–035-003 [HRP]; 1:500) was used against primary goat anti-mouse CD147 antibody (R&D Systems; BAF772; 1:100).

Techniques: Staining, Light Microscopy

Journal: Cell & Bioscience

Article Title: Development of a novel human CD147 knock-in NSG mouse model to test SARS-CoV-2 viral infection

doi: 10.1186/s13578-022-00822-6

Figure Lengend Snippet: Flow cytometric analysis reveals proper dual-expression of both mCD147 and hCD147 in PBMCs and various organs. Representative contour plots of CD147 expression on WT-NSG (top) and hCD147KI het -NSG (bottom) cells from ( A ) PBMCs, ( B ) lung, ( C ) liver, and ( D ) spleen using antibodies targeting either mouse CD147 protein, human CD147 protein, or a combination of both antibodies (far right). Relative percentages are listed, and significant shifts highlighted in red. Gating was determined based on donkey anti-goat/mouse isotype IgG antibody background staining

Article Snippet: To stain mouse CD147 (mCD147), polyclonal donkey anti-goat IgG secondary (Jackson ImmunoResearch; 705–035-003 [HRP]; 1:500) was used against primary goat anti-mouse CD147 antibody (R&D Systems; BAF772; 1:100).

Techniques: Expressing, Staining

Journal: Cell & Bioscience

Article Title: Development of a novel human CD147 knock-in NSG mouse model to test SARS-CoV-2 viral infection

doi: 10.1186/s13578-022-00822-6

Figure Lengend Snippet: Diagram of proposed working hypothesis of CD147 in SARS-CoV-2 infection. (1) SARS-CoV-2 virions infect human cells via the canonical pathway where host Angiotensin-converting Enzyme 2 (ACE2) receptors bind to viral spike proteins (red) and facilitate viral entry and infection. (2) CD147 proteins, via binding to surface binding partners (e.g., E-selectin), facilitate cell–cell adhesion, membrane fusion, and intercellular transfer of SARS-CoV-2 virions. (3) Erythrocytes and platelets which strongly express CD147, bind SARS-CoV-2 virions, and increase thrombosis risk and other clinical manifestations of COVID-19

Article Snippet: To stain mouse CD147 (mCD147), polyclonal donkey anti-goat IgG secondary (Jackson ImmunoResearch; 705–035-003 [HRP]; 1:500) was used against primary goat anti-mouse CD147 antibody (R&D Systems; BAF772; 1:100).

Techniques: Infection, Binding Assay, Membrane

Journal: PLoS ONE

Article Title: Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO

doi: 10.1371/journal.pone.0031564

Figure Lengend Snippet: ( A ) S-nitrosylation of EMMPRIN was detected by the biotin-switch assay in bovine aortic endothelial cells (BAECs). Input refers to EMMPRIN levels in lysates before performing the biotin-switch assay. Specificity of biotinylation was confirmed by incubating samples in the presence or absence of 2 mM ascorbic acid (AA) during the biotin-switch assay. Arrows indicate S-nitrosylated EMMPRIN (EMP). Shown are representative results from 3 independent experiments. ( B ) S-nitrosylated EMMPRIN levels in BAECs in the basal (non-stimulated, left panel) and after eNOS stimulation with 10 µM of a calcium ionophore (A23187) for 30 min to promote NO production (middle panel). Arrow indicates S-nitrosylated EMMPRIN. BAEC lysates immunoprecipitated (IP) with EMMPRIN (EMP, left panel) or S-nitroso-cysteine (SNO-cys, middle panel) antibodies were blotted with SNO-cys or EMMPRIN antibodies, respectively. The bar graph on the right panel shows band intensities of S-nitrosylated EMMPRIN (arrow in the middle panel) in BAECs stimulated with or without A23187. The blots are representative images from 3 independent experiments. ( C ) S-nitrosylation of GOLPH3 was detected by the biotin-switch assay in BAECs. Input refers to GOLPH3 levels in lysates before performing the biotin-switch assay. Specificity of biotinylation was confirmed by incubating samples in the presence or absence of 2 mM ascorbic acid (AA) during the biotin-switch assay. Arrows indicate S-nitrosylated GOLPH3. The blots are representative images from 3 independent experiments. ( D ) S-nitrosylated GOLPH3 levels in BAECs after eNOS stimulation with 10 µM of A23187 for 30 min to promote NO production (left panel). BAEC lysates immunoprecipitated with S-nitrosocysteine (SNO-cys) antibodies were blotted for GOLPH3. The graph on the right panel shows band intensities of S-nitrosylated GOLPH3 (arrow in the left panel) in BAECs stimulated with or without A23187. Shown are representative results from 3 independent experiments.

Article Snippet: The supernatants were collected to new tubes, added 5 μl (1∶100) of S-nitrosocysteine antibody (rabbit, Sigma, St. Louis, MO, Cat#:N5411) or 2 μg of EMMPRIN antibody (goat, Santa Cruz Biotechnology, Santa Cruz, CA, Cat#:sc-9757), and then incubated overnight at 4°C on a rotating mixer.

Techniques: Biotin Switch Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO

doi: 10.1371/journal.pone.0031564

Figure Lengend Snippet: Aorta samples were lysed in a lysis buffer. Three aorta samples were combined per group to obtain a sufficient amount of proteins for the biotin-switch assay. Lysates before the biotin-switch assay were blotted for EMMPRIN and a loading control, heat shock protein 90 (Hsp90) (input, left panel). Equal amounts of proteins (500 µg) in the lysates were used for the biotin-switch assay. Biotinylated proteins were captured by streptavidin agarose beads and blotted with an EMMPRIN antibody (right panel). The aorta from cirrhotic rats showed a higher level of S-nitrosylated EMMPRIN than that of normal rats. Interestingly, only those glycosylated EMMPRIN were S-nitrosylated in the aorta.

Article Snippet: The supernatants were collected to new tubes, added 5 μl (1∶100) of S-nitrosocysteine antibody (rabbit, Sigma, St. Louis, MO, Cat#:N5411) or 2 μg of EMMPRIN antibody (goat, Santa Cruz Biotechnology, Santa Cruz, CA, Cat#:sc-9757), and then incubated overnight at 4°C on a rotating mixer.

Techniques: Lysis, Biotin Switch Assay, Control

Journal: BMC Biotechnology

Article Title: Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells

doi: 10.1186/1472-6750-8-5

Figure Lengend Snippet: Immunocytochemical analysis for colocalization of CD147-intrabody . The transfected 293A cells were fixed and incubated with a mixture of biotinylated anti-human extracellular matrix metalloproteinase inducer (EMMPRIN) mAb and rabbit anti-HA mAb. Then, cells were stained with the mixture of Cy5-conjugated streptavidin and Cy3-conjugated anti-rabbit-IgG mAb. Nuclei were counterstained with DAPI (blue). Three-dimensional (3D) image of the transfected 293A cells was verified. A CD147 on transfected 293A cell stained with biotinylated anti-human EMMPRIN mAb (red), B scFv-M6-1B9 intrabody in transfected 293A cell stained with rabbit anti-HA mAb (white), C GFP positive in transfected 293A cell and D overlay. For 3D image of colocalization of CD147-intrabody see additional file.

Article Snippet: Slides were then washed in PBS containing 50 mM MgCl 2 and blocked with 1% BSA in SSC at RT for 5 min. Then, the fixed cells were incubated with a mixture of biotinylated anti-human extracellular matrix metalloproteinase inducer (EMMPRIN) mAb (0.1 μg/ml; R&D systems, Minneapolis, MN) and rabbit anti-HA mAb (Sigma) at 4°C overnight.

Techniques: Transfection, Incubation, Staining