Journal: Molecular Medicine Reports

Article Title: Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

doi: 10.3892/mmr.2017.6523

Figure Lengend Snippet: SDS-PAGE analysis of the CD147 antigen prior to rabbit immunization. Eukaryotic-expressed recombinant protein CD147-Fc was treated with HRV 3C protease for 16 h, and the mixture was purified by Ni 2+ affinity chromatography followed by Protein A chromatography. SDS-PAGE analysis demonstrated that CD147 was of high purity and indicated that there was no residual Fc fragment or 3C protease. Lane 1, recombinant eukaryotic-expressed CD147-Fc, which has a dimer formation and has been partially degraded; Lane 2, the recombinant CD147-Fc protein was treated with HRV 3C protease for 16 h, resulting in a mixture of CD147-Fc, CD147, Fc fragments and HRV 3C protease; Lane 3, mixture purified by Ni 2+ affinity chromatography, which is not very powerful, and a certain amount of recombinant protein and HRV 3C protease remain; Lane 4, CD147 immunogen purified by Protein A chromatography, no recombinant protein or HRV 3C protease was detected; Lane M, protein molecular weight marker. HRV, human rhinovirus.

Article Snippet: After washing 3 times with PBST, anti-CD147 polyclonal antibodies, which were purified by different methods (including ammonium sulfate precipitation, antigen-immunoaffinity choromatography with ammonium sulfate precipitation and protein A chromatography with ammonium sulfate), or a commercial antibody against CD147 (cat. no. orb42082; Biorbyt Ltd., Cambridge, UK) were added (100 μl/well) and incubated at 37°C for 1 h. After 3 washes with PBST, HRP-conjugated goat anti-rabbit IgG was added (100 μl/well) and the plate was incubated at 37°C for 30 min. After washing 3 times with PBST, TMB substrate was added to the wells (100 μl/well) and was measured at 450 nm using a BioTeck Epoch microplate reader.

Techniques: SDS Page, Recombinant, Purification, Affinity Chromatography, Chromatography, Molecular Weight, Marker

Journal: Molecular Medicine Reports

Article Title: Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

doi: 10.3892/mmr.2017.6523

Figure Lengend Snippet: SDS-PAGE and western blot analysis of CD147-GST fusion protein purification and identification. The prokaryotic-expressed CD147-GST fusion protein was purified by a GSTrap column. The purified protein had a high purity. (A) SDS-PAGE analysis of purified CD147-GST. Lane 1, supernatant of the lysate of CD147-GST; Lane 2, flow through liquid of the supernatant; Lane 3, flow through liquid of the washing buffer; Lane 4, protein eluted from the column; Lane M, protein molecular weight marker. (B) Western blot analysis of purified CD147-GST. The primary antibody was HAb18, which has high specificity and affinity for CD147. The secondary antibody was goat anti-mouse immunoglobulin G conjugated with horseradish peroxidase. Western blotting confirmed that the observed band corresponded to CD147-GST protein. GST, glutathione S-transferase.

Article Snippet: After washing 3 times with PBST, anti-CD147 polyclonal antibodies, which were purified by different methods (including ammonium sulfate precipitation, antigen-immunoaffinity choromatography with ammonium sulfate precipitation and protein A chromatography with ammonium sulfate), or a commercial antibody against CD147 (cat. no. orb42082; Biorbyt Ltd., Cambridge, UK) were added (100 μl/well) and incubated at 37°C for 1 h. After 3 washes with PBST, HRP-conjugated goat anti-rabbit IgG was added (100 μl/well) and the plate was incubated at 37°C for 30 min. After washing 3 times with PBST, TMB substrate was added to the wells (100 μl/well) and was measured at 450 nm using a BioTeck Epoch microplate reader.

Techniques: SDS Page, Western Blot, Protein Purification, Purification, Molecular Weight, Marker

Journal: Molecular Medicine Reports

Article Title: Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

doi: 10.3892/mmr.2017.6523

Figure Lengend Snippet: Characterization of the purified anti-CD147 polyclonal antibody. (A) SDS-PAGE analysis of purified polyclonal antibody. The rabbit antiserum against CD147 was purified by ammonium sulfate precipitation followed by antigen-immunoaffinity chromatography. Lane M, protein molecular weight marker; Lane 1, polyclonal antibody purified by antigen-immunoaffinity chromatography; Lane 2, washing liquid of antigen-immunoaffinity chromatography; Lane 3, flow liquid of antigen-immunoaffinity chromatography; Lane 4, polyclonal antibody purified by ammonium sulfate precipitation only; Lane 5, antiserum against CD147. (B) The reactivity of the antibodies purified by antigen-immunoaffinity chromatography was determined by indirect ELISA. The results demonstrated that the purified antibody had a high reactivity.

Article Snippet: After washing 3 times with PBST, anti-CD147 polyclonal antibodies, which were purified by different methods (including ammonium sulfate precipitation, antigen-immunoaffinity choromatography with ammonium sulfate precipitation and protein A chromatography with ammonium sulfate), or a commercial antibody against CD147 (cat. no. orb42082; Biorbyt Ltd., Cambridge, UK) were added (100 μl/well) and incubated at 37°C for 1 h. After 3 washes with PBST, HRP-conjugated goat anti-rabbit IgG was added (100 μl/well) and the plate was incubated at 37°C for 30 min. After washing 3 times with PBST, TMB substrate was added to the wells (100 μl/well) and was measured at 450 nm using a BioTeck Epoch microplate reader.

Techniques: Purification, SDS Page, Chromatography, Molecular Weight, Marker, Indirect ELISA

Journal: Molecular Medicine Reports

Article Title: Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

doi: 10.3892/mmr.2017.6523

Figure Lengend Snippet: Sensitivity test results of human CD147 sandwich ELISA.

Article Snippet: After washing 3 times with PBST, anti-CD147 polyclonal antibodies, which were purified by different methods (including ammonium sulfate precipitation, antigen-immunoaffinity choromatography with ammonium sulfate precipitation and protein A chromatography with ammonium sulfate), or a commercial antibody against CD147 (cat. no. orb42082; Biorbyt Ltd., Cambridge, UK) were added (100 μl/well) and incubated at 37°C for 1 h. After 3 washes with PBST, HRP-conjugated goat anti-rabbit IgG was added (100 μl/well) and the plate was incubated at 37°C for 30 min. After washing 3 times with PBST, TMB substrate was added to the wells (100 μl/well) and was measured at 450 nm using a BioTeck Epoch microplate reader.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Molecular Medicine Reports

Article Title: Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

doi: 10.3892/mmr.2017.6523

Figure Lengend Snippet: Comparison of polyclonal antibodies against CD147 purified by antigen-immunoaffinity chromatography and other methods of purification.

Article Snippet: After washing 3 times with PBST, anti-CD147 polyclonal antibodies, which were purified by different methods (including ammonium sulfate precipitation, antigen-immunoaffinity choromatography with ammonium sulfate precipitation and protein A chromatography with ammonium sulfate), or a commercial antibody against CD147 (cat. no. orb42082; Biorbyt Ltd., Cambridge, UK) were added (100 μl/well) and incubated at 37°C for 1 h. After 3 washes with PBST, HRP-conjugated goat anti-rabbit IgG was added (100 μl/well) and the plate was incubated at 37°C for 30 min. After washing 3 times with PBST, TMB substrate was added to the wells (100 μl/well) and was measured at 450 nm using a BioTeck Epoch microplate reader.

Techniques: Comparison, Purification, Chromatography, Sandwich ELISA, Concentration Assay, Affinity Chromatography

Journal: Oncology Letters

Article Title: Expression and clinical significance of extracellular matrix metalloproteinase inducer, EMMPRIN/CD147, in human osteosarcoma

doi: 10.3892/ol.2012.981

Figure Lengend Snippet: Correlation between EMMPRIN/CD147 expression and clinicopathological characteristics.

Article Snippet: The sections were blocked with 5% bovine serum albumin (Zhongshan, Beijing, China) in PBS solution for 20 min and probed with rabbit anti-human EMMPRIN/CD147 antibodies (1:300; Boster) at 4°C for 12 h. After washing, the bound antibodies were detected with biotinylated goat anti-rabbit IgG (1:100) and SABC complex at 30°C for 20 min.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Active Vaccination With EMMPRIN-Derived Multiple Antigenic Peptide (161-MAP) Reduces Angiogenesis in a Dextran Sodium Sulfate (DSS)-Induced Colitis Model

doi: 10.3389/fimmu.2018.02919

Figure Lengend Snippet: 161-MAP active vaccination reduces EMMPRIN expression in the colon. (A) Representative images of colon sections stained for EMMPRIN and their quantification using the H-score ( n = 5 per group). Scale bar is 25 μm (B) Representative image of fluorescently labeled EMMPRIN (red) and macrophages (green). Scale bar is 100 μm. (C) Determination of EMMPRIN concentrations in colon lysates ( n = 9–10 per group), and in serum samples ( n = 8–9 per group) by ELISA.

Article Snippet: Endogenous peroxidase was quenched in 3% H 2 O 2 solution for 10 min, slides were blocked with 5% BSA and incubated overnight at 4°C with the following primary antibodies: rat anti-mouse EMMPRIN (R&D systems, MAB772, Minneapolis, MN, USA) diluted 1:250; rat monoclonal anti-F4/80 (Abcam, ab6640, Cambridge, UK) diluted 1:200; rabbit polyclonal anti-CD8 (Bioss, bs-0648R, Woburn, MA, USA) diluted 1:400.

Techniques: Expressing, Staining, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Active Vaccination With EMMPRIN-Derived Multiple Antigenic Peptide (161-MAP) Reduces Angiogenesis in a Dextran Sodium Sulfate (DSS)-Induced Colitis Model

doi: 10.3389/fimmu.2018.02919

Figure Lengend Snippet: 161-MAP active vaccination reduces angiogenesis. (A) Colon sections were stained for CD31 and the vessel surface area was calculated ( n = 4 per group). Scale bar is 100 μm. (B) Concentrations of VEGF and MMP-9 were determined in serum sample by ELISA, and in the colon lysates, normalized to the total protein amounts ( n = 9 per group). (C) Wound scratch assay: colon lysates (25 μg of total protein) were diluted (1:4) and applied onto a confluent layer of the mouse bEND3 endothelial cells (10 5 cells/ 96-plate well) that was scratched with a toothpick. Images were acquired at the beginning of the experiment (T0) and at the end after 24h (T24). The migration area was calculated by subtracting the area of the wound at T24, after endothelial cell migrated and partially closed the wound, from the area of the wound at T0. An EMMPRIN specific blocking antibody (161-pAb) was added to some of the wells as indicated. ( n = 9–10 for the male mice, n = 8 for the female mice). Magnification is x4.

Article Snippet: Endogenous peroxidase was quenched in 3% H 2 O 2 solution for 10 min, slides were blocked with 5% BSA and incubated overnight at 4°C with the following primary antibodies: rat anti-mouse EMMPRIN (R&D systems, MAB772, Minneapolis, MN, USA) diluted 1:250; rat monoclonal anti-F4/80 (Abcam, ab6640, Cambridge, UK) diluted 1:200; rabbit polyclonal anti-CD8 (Bioss, bs-0648R, Woburn, MA, USA) diluted 1:400.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Migration, Blocking Assay

Journal: IUBMB life

Article Title: CD147 regulates vascular endothelial growth factor-A expression, tumorigenicity, and chemosensitivity to curcumin in hepatocellular carcinoma.

doi: 10.1002/iub.11

Figure Lengend Snippet: Figure 1. Silencing of CD147 downregulates VEGF-A expression in Hepa1-6 cells by RNAi approach. The Hepa1-6 cells were transfected with CD147 siRNA and control siRNA. Total RNA was extracted and analyzed by RT-PCR using primers specific to CD147, VEGF-A and GAPDH (A). Cells collected on the indicated day (RNAi-3, 3 days; RNAi-7, 7 days) were lysed, and the expressions of CD147 and VEGF-A were analyzed by western-blot analysis using anti-Axl or anti-VEGF-A antibodies (B). GAPDH was also examined and served as controls for sample loading. (C) CD147 expression did not recover to original level until 12 days after siRNA transfection by RT-PCR.

Article Snippet: Rabbit anti-mouse CD147 polyclonal antibody (1:50 dilution, Santa Cruz Biotech) was used in standard indirect immunoperoxidase procedures.

Techniques: Expressing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: IUBMB life

Article Title: CD147 regulates vascular endothelial growth factor-A expression, tumorigenicity, and chemosensitivity to curcumin in hepatocellular carcinoma.

doi: 10.1002/iub.11

Figure Lengend Snippet: Figure 2. Silencing of CD147 impedes Hepa1-6 cells proliferation and anchorage-independent growth in vitro. A: Cells were har- vested at 24, 48, 72, 96, and 120 hours posttransfection, and cell proliferation was measured by MTT assay. Decreased growth abil- ity was detected in CD147/RNAi cells (RNAi-3, 3 days; RNAi-7, 7 days) compared with RNAi control cells. B: Colony-forming activities of CD147/RNAi cells in soft agar. The cell colonies were photographed (3100). C: Colony formation numbers were com- pared between transfected and control RNAi cells. Significant downregulation of colony formation numbers was confirmed in CD147/RNAi cells compared with control RNAi cells (*p \ 0.05). The data were obtained from three independent experiments.

Article Snippet: Rabbit anti-mouse CD147 polyclonal antibody (1:50 dilution, Santa Cruz Biotech) was used in standard indirect immunoperoxidase procedures.

Techniques: In Vitro, MTT Assay, Control, Transfection

Journal: IUBMB life

Article Title: CD147 regulates vascular endothelial growth factor-A expression, tumorigenicity, and chemosensitivity to curcumin in hepatocellular carcinoma.

doi: 10.1002/iub.11

Figure Lengend Snippet: Figure 3. Silencing of CD147 in the Hepa1-6 cells inhibits tumorigenicity in nude mice. A: Four groups of nude mice were injected subcutaneously with Hepa1-6, RNAi control, and CD147/RNAi cells (RNAi-3, 3 days; RNAi-7, 7 days). After 3 weeks, tumors were excised, weighed, sized and photographed. B and C: CD147/RNAi groups showed a significant decrease in mean tu- mor weight (n 5 10) and volume (n 5 10) (*p \ 0.05), compared with control groups. D: Histological analysis was performed in implanted tumors generated from Hepa1-6, RNAi control, and CD147/RNAi cells with H&E staining (RNAi-3, 3 days; RNAi-7, 7 days), respectively, 3400. E: CD147 protein expression was determined by immunohistochemistry staining in solid tumors derived from Hepa1-6, RNAi control and CD147/RNAi cells (3400). Differences in CD147 protein expression were shown. The data were obtained from three independent experiments.

Article Snippet: Rabbit anti-mouse CD147 polyclonal antibody (1:50 dilution, Santa Cruz Biotech) was used in standard indirect immunoperoxidase procedures.

Techniques: Injection, Control, Generated, Staining, Expressing, Immunohistochemistry, Derivative Assay

Journal: IUBMB life

Article Title: CD147 regulates vascular endothelial growth factor-A expression, tumorigenicity, and chemosensitivity to curcumin in hepatocellular carcinoma.

doi: 10.1002/iub.11

Figure Lengend Snippet: Figure 4. Silencing of CD147 in the Hepa1-6 cells increases the chemosensitivity to curcumin. Hepa1-6, RNAi control, and CD147/RNAi cells (RNAi-3, 3 days; RNAi-7, 7 days) were treated with various concentrations of curcumin. Cell viability was determined by MTT chronometry. Cell survival rate (%) was calculated as A570 (curcumin1)/A570 (curcumin2) 3 100%. The data were obtained from three independent experiments (*p \ 0.05).

Article Snippet: Rabbit anti-mouse CD147 polyclonal antibody (1:50 dilution, Santa Cruz Biotech) was used in standard indirect immunoperoxidase procedures.

Techniques: Control

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