Journal: OncoImmunology

Article Title: An epitope-specific novel anti-EMMPRIN polyclonal antibody inhibits tumor progression

doi: 10.1080/2162402x.2015.1078056

Figure Lengend Snippet: Figure 1: EMMPRIN structure and peptide design, and specificity of 161-Ab: (A) Partial

Article Snippet: One strip was probed with the 1:1,000 diluted commercial rat anti-mouse EMMPRIN (MAB772, R&D systems) and then with the 1:5,000 diluted HRP-conjugated goat anti-rat IgG (112-035- D ow nl oa de d by [ U ni ve rs ity o f R eg in a] a t 0 6: 58 2 4 Fe br ua ry 2 01 6 062, Jackson) and served as positive control (P.C).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

doi: 10.1111/j.1582-4934.2010.01012.x

Figure Lengend Snippet: Measurement of HAb18G/CD147 expression on resting and activated CD 24+ and CD 8+ T cell subsets. Purified resting T cells and activated T cells were stained with PE- conjugated anti-CD147, FITC-conjugated anti-CD4 or CD8, and were analysed by flow cytometry. (A) Histogram shows expression of CD147 on resting and activated CD 24+ T cells. (B) Histogram shows expression of CD147 on resting and activated CD 8+ T cells. These data are representative of a minimum of three separate experiments.

Article Snippet: RPE-conjugated mouse anti-human CD147 was obtained from AbD Serotec (Oxford, UK).

Techniques: Expressing, Purification, Staining, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

doi: 10.1111/j.1582-4934.2010.01012.x

Figure Lengend Snippet: HAb18G/CD147 accumulates to the cap and lipid rafts in T cells upon TCR stimulation confirmed by confocal microscopy and Western blot. (A, B, C, D) HAb18G/CD147 co-caps with CD4/CD8/CD48/GM1. T cells were prepared and stained as described previously in ‘Materials and methods’. HAb18G/CD147, TCR co-receptor CD4 and CD8, lipid raft marker CD48 and GM1 diffusely distribute on the cell membrane of resting T cells (upper panel of –D), whereas stimulation results in translocation of these molecules to the cap induced by TCR/CD3 (lower panel of –D), scale bar, 5 μm. (E) HAb18G/CD147 is recruited to lipid rafts upon T cell activation. Lipid rafts were isolated as described previously in ‘Materials and methods’. Sucrose fractions were analysed by Western blot with HAb18, anti-Fyn and anti-Rab5 antibodies. Detergent-insoluble membrane fractions containing isolated rafts were identified by the presence of the raft marker protein Fyn. Non-raft components of the membrane ( e.g. Rab5) were purified in the heavy fraction (HF). HAb18G/CD147 appeared constitutively in the HF (7–9) with the non-raft marker Rab5 in resting T cells, a significant fraction of HAb18G/CD147 was localized within raft fractions (1–4) after T cell activation.

Article Snippet: RPE-conjugated mouse anti-human CD147 was obtained from AbD Serotec (Oxford, UK).

Techniques: Confocal Microscopy, Western Blot, Staining, Marker, Membrane, Translocation Assay, Activation Assay, Isolation, Purification

Journal: Journal of Cellular and Molecular Medicine

Article Title: Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

doi: 10.1111/j.1582-4934.2010.01012.x

Figure Lengend Snippet: HAb18G/CD147 together with CD48 and GM1 are enriched into the IS. Raji cells were labelled with the blue fluorescent cytoplasmic probe CMAC first. Jurkat cells were then incubated with CMAC-labelled and 1 μg/ml of SEB-loaded (or not) Raji cells. Conjugates were then plated onto cover slip, fixed, stained with antibodies to HAb18G/CD147 and to CD48 or GM1 , and visualized by confocal fluorescence microscopy. The red and green images represent the localization and distribution of HAb18G/CD147 and CD48/GM1, respectively. Raji cells are shown in blue. Scale bar, 5 μm.

Article Snippet: RPE-conjugated mouse anti-human CD147 was obtained from AbD Serotec (Oxford, UK).

Techniques: Incubation, Staining, Fluorescence, Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

doi: 10.1111/j.1582-4934.2010.01012.x

Figure Lengend Snippet: Influence of HAb18G/CD147 mAbs on T cell proliferation. Proliferation was determined on day 4 following stimulation as described in ‘Materials and methods’. This figure shows the [ 3 H]-thymidine incorporation in c.p.m. (mean ± S.D. of triplicated wells). Data are a representative of three independent experiments. ***, P , 0.001.

Article Snippet: RPE-conjugated mouse anti-human CD147 was obtained from AbD Serotec (Oxford, UK).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

doi: 10.1111/j.1582-4934.2010.01012.x

Figure Lengend Snippet: Cross-linking of HAb18G/CD147 with mAb 5A12 affects surface expression of CD25 and cytokine production. (A) mAb 5A12 down-regulates surface expression of CD25. Red-tinted histogram represents isotype control. Black and green lines represent anti-CD3 mAb plus isotype-matched irrelevant control mAb (mouse IgG1) or 5A12 intervention, respectively. (B, C, D) mAb 5A12 decreases IL-2 and IL-4 but increases IFN-γ production. Purified T cells were stimulated with immobilized 1 μg/ml of anti-CD3 mAb or 1 μg/ml of anti-CD3 mAb plus 10 μg/ml anti-CD28 mAb in the presence of 10 μg/ml mAb 5A12 or isotype-matched irrelevant control for 72 hrs. Bar graphs, (mean ± S.D.) levels of IL-2, IL-4 and IFN-γ in culture supernatants as measured by ELISA. *, P , 0.05.

Article Snippet: RPE-conjugated mouse anti-human CD147 was obtained from AbD Serotec (Oxford, UK).

Techniques: Expressing, Control, Purification, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

doi: 10.1111/j.1582-4934.2010.01012.x

Figure Lengend Snippet: Inhibition of anti-CD3 stimulated redistribution of the CD48 molecule by HAb18G/CD147 mAb 5A12. Purified peripheral blood T cells were cultured and stimulated with immobilized CD3 mAb in the presence or absence of 10 μg/ml of HAb18G/CD147 mAb 5A12 or isotype-matched irrelevant control mAb for 2 hrs. Then, cells were collected, fixed and stained with FITC-labelled CD48 as described in ‘Materials and methods’. The percentage of cells showing cap was assessed by analysing the distribution of the fluorescence intensity on the cell membrane of CD48-FITC stained cells using FV10-ASW (version 1.6) software. The images are a representative of three experiments. Scale bar, 5 μm. **, P , 0.01.

Article Snippet: RPE-conjugated mouse anti-human CD147 was obtained from AbD Serotec (Oxford, UK).

Techniques: Inhibition, Purification, Cell Culture, Control, Staining, Fluorescence, Membrane, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

doi: 10.1111/j.1582-4934.2010.01012.x

Figure Lengend Snippet: Cross-linking of HAb18G/CD147 with mAb 5A12 affects intracellular signalling pathway upon T cell activation. (A) Extracellular Ca 2+ influx triggered by TCR stimulation is inhibited by mAb 5A12. The figure is a representative of three experiments. (B) HAb18G/CD147 mAb 5A12 slightly decreases the tyrosine phosphorylation level upon TCR triggering. Purified T cells were stimulated using CD3 mAb (plus mAb CD28) in the presence of mAb 5A12 or isotype-matched irrelevant control. After 2 hrs of stimulation, the cells were lysed and analysed for tyrosine phosphorylation level using Western blot. The figure is a representative of three experiments. Top, the representative image; bottom, quantitative analysis of the image.

Article Snippet: RPE-conjugated mouse anti-human CD147 was obtained from AbD Serotec (Oxford, UK).

Techniques: Activation Assay, Phospho-proteomics, Purification, Control, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

doi: 10.1111/j.1582-4934.2010.01012.x

Figure Lengend Snippet: Computer-assisted molecular docking of HAb18G/CD147 antigen-antibody complex. (A) The modelled structure of the variable domain of mAb HAb18 was docked to the most membrane-distal region of HAb18G/CD147 in a head-to-head manner. (B) The variable domain of mAb 6H8 was docked to C-terminal domain II of HAb18G/CD147. (C) The interaction between mAb 5A12 and HAb18G/CD147. The binding zone localizes at the functional N-terminal domain I. (D) Overview of docking the three mAbs to HAb18G/CD147 extracellular portion.

Article Snippet: RPE-conjugated mouse anti-human CD147 was obtained from AbD Serotec (Oxford, UK).

Techniques: Membrane, Binding Assay, Functional Assay

Journal: Cells

Article Title: SARS-CoV-2 Entry: At the Crossroads of CD147 and ACE2

doi: 10.3390/cells10061434

Figure Lengend Snippet: Ordering information and working conditions of used antibodies.

Article Snippet: CaLu3 cells were treated with 1 μg/mL anti-CD147 antibody (MEM-M6/6, Bio-rad Laboratories, Hercules, CA, USA) or IgG of mouse origin (015-000-003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted in complete medium 1 h before and during infection.

Techniques:

Journal: Cells

Article Title: SARS-CoV-2 Entry: At the Crossroads of CD147 and ACE2

doi: 10.3390/cells10061434

Figure Lengend Snippet: Inhibition of CyPA binding to CD147 does not play a role in SARS-CoV-2 entry. CaLu3 cells were treated with 1 µg/mL MEM-M6/6 CD147 blocking Ab (α-CD147 Ab) or equivalent mouse IgG (as isotype control) or plain medium (as control, ctr) for one hour before, and during SARS-CoV-2 infection. Samples were collected 48 hpi. ( A ) N1 RNA levels were analyzed by ddPCR and their relative abundance in cells is shown. N1 RNA levels were normalized to total RNA. N1 levels in CD147-blocking antibody treated samples were normalized to the levels in the corresponding IgG isotype-treated control. Mean ± s.e.m out of three biological replicates is shown. n.s. not significative, Student’s t test. ( B , C ) Lysates were analyzed by WB. ( B ) One experiment is shown as representative of three. Immunolabelling for CD147 showed that Ab treatment did not affect the levels of expression of the receptor either in the absence or in the presence of SARS-CoV-2. ( C ) Spike protein levels were normalized to GAPDH (loading control). Spike levels in CD147-blocking antibody treated samples were normalized to the levels in the corresponding IgG isotype-treated control. Mean ± s.e.m. out of three biological replicates is shown. n.s. not significant, Student’s t -test.

Article Snippet: CaLu3 cells were treated with 1 μg/mL anti-CD147 antibody (MEM-M6/6, Bio-rad Laboratories, Hercules, CA, USA) or IgG of mouse origin (015-000-003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted in complete medium 1 h before and during infection.

Techniques: Inhibition, Binding Assay, Blocking Assay, Control, Infection, Expressing

Journal: Cells

Article Title: SARS-CoV-2 Entry: At the Crossroads of CD147 and ACE2

doi: 10.3390/cells10061434

Figure Lengend Snippet: CD147 silencing reduces ACE2 protein levels and impairs SARS-CoV-2 infection. ( A – C ) CaLu3 cells were transfected with a non-targeting (NT) or a CD147-specific siRNA. Samples were collected 48 h and 96 h after transfection. ( A ) One experiment is shown as representative of three. ( B , C ) Receptor levels were analysed by WB (protein, ( B )) and by ddPCR (RNA, ( C )) analysis. ( B ) Receptors’ protein levels were normalized to the loading control (GAPDH). Receptors levels in CD147 siRNA-treated samples were normalized to the levels in the corresponding NT siRNA-treated controls. Mean ± s.e.m. out of three biological replicates is shown. **, p = 0.005 for CD147 and p = 0.01 for ACE2, Student’s t -test. ( C ) Receptors RNA levels were normalized to total RNA. Receptors levels in CD147 siRNA-treated samples were normalized to the levels in the corresponding NT siRNA-treated controls. Mean ± s.e.m. out of three biological replicates is shown. ***, p = 0.001, n.s. not significative, Student’s t -test. ( D , E ) CaLu3 cells were transfected with a non-targeting (NT) or a CD147-specific siRNA before SARS-CoV-2 infection. Samples were collected 48 hpi. ( D ) N1 RNA levels were analyzed by ddPCR, and their relative abundance in both cells and supernatants is shown. N1 RNA levels were normalized to total RNA. N1 levels in CD147 siRNA-treated samples were normalized to the levels in the corresponding NT siRNA-treated controls. Mean ± s.e.m. out of three biological replicates is shown. **, p < 0.01; *, p < 0.05, Student’s t -test. ( E ) Lysates were analyzed by WB. One experiment is shown as representative of three. ACE2 labeling was detected after Spike protein immunostaining on the same membrane. Arrow indicates ACE2 specific band. GAPDH was used as the loading control.

Article Snippet: CaLu3 cells were treated with 1 μg/mL anti-CD147 antibody (MEM-M6/6, Bio-rad Laboratories, Hercules, CA, USA) or IgG of mouse origin (015-000-003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted in complete medium 1 h before and during infection.

Techniques: Infection, Transfection, Control, Labeling, Immunostaining, Membrane

Journal: Cells

Article Title: SARS-CoV-2 Entry: At the Crossroads of CD147 and ACE2

doi: 10.3390/cells10061434

Figure Lengend Snippet: CD147 and ACE2 expression is affected by SARS-CoV-2 infection. ( A , B ) CaLu3 cells were infected with SARS-CoV-2 at 0.05 MOI and samples collected 48 hpi. Receptors levels were analyzed by ddPCR (RNA, ( A )) and WB (protein, ( B )). ( A ) Receptors RNA levels were normalized to total RNA. Receptors levels in infected samples (+SARS-CoV-2) were normalized to the levels in the corresponding uninfected controls (-SARS-CoV-2). Mean ± s.e.m. out of three biological replicates is shown. *, p = 0.02; ***, p = 0.001, Student’s t -test. ( B ) Receptor protein levels were normalized to the loading control. Receptor levels in infected samples (+SARS-CoV-2) were normalized to the levels in the corresponding uninfected controls (-SARS-CoV-2). Mean ± s.e.m. out of 10 biological replicates is shown. ***, p < 0.0001, Student’s t -test.

Article Snippet: CaLu3 cells were treated with 1 μg/mL anti-CD147 antibody (MEM-M6/6, Bio-rad Laboratories, Hercules, CA, USA) or IgG of mouse origin (015-000-003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted in complete medium 1 h before and during infection.

Techniques: Expressing, Infection, Control