Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: Fluorescent micrographs of HEK293 cells transfected with (A, B) SAM-6 μHC and SAM-6 λLC construct pair, (C, D) SAM-6 μHC subunit alone, or (E, F) SAM-6 λLC subunit alone. On day-2 post-transfection, transfected cells were seeded onto poly-D-lysine coated coverslips in the absence (A, C, E) or presence (B, D, F) of 15 μg/mL Brefeldin A (BFA) and cultured statically for 24 hr. On day-3 post-transfection, cells were fixed, permeabilized, and immunostained. (A, B) Co-staining was performed using FITC-labeled anti-human μHC antibody and Texas Red-labeled anti-human λLC antibody. (C, D) Co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. (E, F) Co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human λLC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to make ‘overlay’ views in A and B. DIC and red image fields were superimposed to create ‘overlay’ views in C‒F.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Transfection, Construct, Cell Culture, Staining, Labeling

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A) Schematic representation of SAM-6 λLC subunit (top), SAM-6 μHC subunit (middle), and SAM-6 μHC-ΔCH1 mutant which lacks the CH1 domain (bottom). The deleted CH1 domain is shown as a dotted line. Individual domain names are indicated in each box. ER targeting is driven by a heterologous signal sequence adapted from a VK1 encoding gene. (B) Fluorescent micrographs of HEK293 cells transfected with μHC (first row) or μHC-ΔCH1 mutant (second and third rows). On day-3 post-transfection, cells were fixed, permeabilized, and immunostained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views. (C) Fluorescent micrographs of HEK293 cells transfected with [λLC + μHC] pair (first row) or [λLC + μHC-ΔCH1] pair (second and third rows). Immunostaining was performed using FITC-labeled anti-human μHC antibody and Texas Red-labeled anti-human λLC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to create ‘overlay’ views. (D‒G) HEK293 cells were transfected with μHC (lanes 1 and 5), μHC-ΔCH1 (lanes 2 and 6), [λLC + μHC] pair (lanes 3 and 7), or [λLC + μHC-ΔCH1] pair (lanes 4 and 8). Cell culture media were harvested at day-7 post-transfection and analyzed by SDS-PAGE under reducing conditions (D, E; lanes 1‒4) or non-reducing conditions (F, G; lanes 1‒4). Cell lysate samples were also prepared on day-7 post-transfection and analyzed by SDS-PAGE (D, lanes 5‒8) or Western blotting (E, lanes 5‒8) after resolving the proteins under reducing conditions. Western blotting was performed using polyclonal anti-IgM (H+L) to detect both μHC and λLC subunits simultaneously as well as assembly intermediates composed of μHC or λLC or both. A faintly detectable μHC-ΔCH1 is pointed by a black arrowhead (E, lane 4). Likewise, faintly detectable μHC-ΔCH1 covalent dimers are pointed by black arrowhead (G, lane 4). The assembled hexameric IgM product is pointed by a red arrowhead (F, G; lane 3). Identifiable assembly intermediates are labeled next to the corresponding bands in panels F and G.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Mutagenesis, Sequencing, Transfection, Labeling, Immunostaining, Cell Culture, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A, top) Schematic representation of the full-length SAM-6 λLC (top row) and its ΔCS mutant (second row) in which two C-terminal amino acids (Cys-213 and Ser-214) are deleted. (A, bottom) The position of Cys-213 residue involved in the HC‒LC inter-chain disulfide bond is highlighted in yellow in the context of the hexameric IgM diagram. Solid red lines represent the inter-chain disulfide bond connectivity. (B, C) HEK293 cells were transfected with full-length λLC (lanes 1, 3, 5) or its ΔCS mutant (lanes 2, 4, 6). At day-7 post-transfection, cell lysates (lanes 1 and 2) and cell culture media samples (lanes 3 and 4) were prepared and resolved by SDS-PAGE under reducing conditions followed by Coomassie blue staining (B) or by Western blotting (C). The day-7 cell culture media were also analyzed by Coomassie staining or Western blotting after resolving the proteins under non-reducing conditions (B and C, lanes 5 and 6). Blotted membranes were probed with polyclonal anti-λLC antibodies. The corresponding protein band for the λLC subunit is pointed by an arrowhead and labeled (lanes 1‒4). Monomeric and dimeric free λLC subunit is labeled next to lane 6. (D, E) Fluorescent micrographs of HEK293 cells transfected with full-length λLC (D) or ΔCS mutant (E). On day-3 post-transfection, cells were fixed, permeabilized, and co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human λLC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Mutagenesis, Residue, Transfection, Cell Culture, SDS Page, Staining, Western Blot, Labeling

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A, left) Schematic representation of parental SAM-6 μHC (top row) and its C137S and C337S mutants (second and third rows). (A, right) The position of Cys-137 and Cys-337 residues is highlighted in yellow in the context of the hexameric IgM diagram. Solid red lines represent the inter-chain disulfide bond connectivity. (B, C) HEK293 cells were transfected with parental μHC and its mutants, as shown at the top of each lane. At day-7 post-transfection, cell culture media (B) and cell lysates (C) were prepared and resolved by SDS-PAGE under reducing conditions followed by Coomassie blue staining (B, C, left panels) or by Western blotting (B, C, right panels). Blotted membranes in B and C were probed with polyclonal anti-IgM (H+L) antibodies. Both parental and mutant μHCs failed to secrete to the culture media. (D) Fluorescent micrographs of HEK293 cells transfected with parental μHC (top row), μHC (C137S) mutant (second and third rows), or μHC (C337S) mutant (fourth and fifth rows). On day-3 post-transfection, cells were fixed, permeabilized, and co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Transfection, Cell Culture, SDS Page, Staining, Western Blot, Mutagenesis, Labeling

Journal: bioRxiv

Article Title: Understanding the biosynthesis of human IgMs through a combinatorial expression of mutant subunits that affect different assembly steps

doi: 10.1101/2023.09.01.555973

Figure Lengend Snippet: (A) Schematic representation of parental SAM-6 μHC (top row) and its 4×C>S mutant (second row). The positions of key cysteine residues important for the inter-chain disulfide formation are marked on the parental μHC. (B) The position of all four Cys residues involved in inter-chain disulfide bond formation on μHC and Cys-213 on λLC are depicted in the context of the hexameric IgM diagram. Solid red lines represent the inter-chain disulfide bond connectivity. (C, D) HEK293 cells were transfected with parental μHC alone (lane 1) or its 4×C>S mutant alone (lane 2). Likewise, the cells are co-transfected with μHC and JC (lane 3) or μHC (4×C>S) and JC (lane 4). At day-7 post-transfection, cell lysates (C) and culture media (D) were resolved by SDS-PAGE under reducing conditions followed by Coomassie blue staining (C, D; left panel) or by Western blotting (C, D; right panels). Blotted membranes in C and D were probed with polyclonal anti-IgM (H+L) antibody (top panel) or monoclonal anti-JC antibody (bottom panel). (E) Day-7 culture media were also analyzed by Western blotting after proteins were resolved under non-reducing conditions. Blotted membranes were probed with polyclonal anti-IgM (H+L) antibody (left panel) or monoclonal anti-JC antibody (right panel). (F) Fluorescent micrographs of HEK293 cells transfected with parental μHC (top row) or μHC (4×C>S) mutant (second and third rows). On day-3 post-transfection, cells were fixed, permeabilized, and co-stained with FITC-labeled anti-CD147 antibody and Texas Red-labeled anti-human μHC antibody. Green and red image fields were superimposed to create ‘merge’ views. DIC and red image fields were superimposed to create ‘overlay’ views. (G) Fluorescent micrographs of HEK293 cells co-transfected with μHC and JC (top row) or μHC (4×C>S) mutant and JC (second and third rows). Cells were co-stained FITC-labeled anti-human μHC (shown in green) and monoclonal anti-JC antibody followed by AlexaFluor594-conjugated secondary antibody (shown in red). Green and red image fields were superimposed to create ‘merge’ views. DIC and ‘merge’ were superimposed to create ‘overlay’ views.

Article Snippet: FITC-conjugated mouse monoclonal anti-CD147 (clone HIM6) (cat. 555962) was obtained from BD Biosciences.

Techniques: Mutagenesis, Transfection, SDS Page, Staining, Western Blot, Labeling

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: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 1. Validation of the CT26-KD cells. The parental CT26 cells (WT) and knocked-down CT26 cells (KD) were seeded (8 × 104 cells each) in 24-well plates in 400 µL full medium for 48 h. At the end of the incubation, (A) total RNA was extracted from the cells and amplified using EMMPRIN specific primers (n = 4), and (B) the supernatants were collected for an ELISA analysis of EMMPRIN secretion (n = 9). Data are presented as means ± SE and analyzed using the two-tailed Student’s t test analysis. (C) CT26-WT and CT26-KD cells (30,000 cells/well/300 µL) were stained as described in the methods. A representative image, demonstrating reduced EMMPRIN protein expression in the CT26-KD cells (n = 3). Bar size is 20 µM. (D) EMMPRIN is known to appear in several bands, reflecting its low and high glycosylation patterns. Western blot analysis demonstrates that in the CT26-KD cells, all EMMPRIN bands showed a reduced expression of EMMPRIN.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Biomarker Discovery, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Expressing, Glycoproteomics, Western Blot

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 2. Co-culturing enhances the secretion of EMT-driver cytokines. CT26-WT or CT26-KD cells (80,000 cells each) were each cultured alone or co-cultured with RAW 264.7 cells that were seeded in the upper chamber of the inserts (0.4 µm pore size) at a ratio of 1:1, in serum-starvation medium (final volume 650 µL) for 48 h. At the end of the incubation, supernatants were collected and the concentrations of (A) TGFβ (n = 5), (B) soluble EMMPRIN (n = 6), (C) TNFα (n = 6), and (D) IL-6 (n = 6) were determined by ELISA. Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Cell Culture, Pore Size, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 4. Expression of the EMT-TFs and dormancy markers is enhanced in the CT26-KD cells, but reduced in the co-culture or its simulation. CT26-WT or CT26-KD cells (8 × 104 cells each) were incubated alone or in co-culture with RAW 264.7 cells as described before for 48 h. Alternatively, single cultures of CT26-WT or CT26-KD cells (8 × 104 cells) were cultured with or without the addition of recombinant TGFβ (10 ng/mL) or recombinant EMMPRIN (25 ng/mL or 250 ng/mL). Total RNA was extracted from the CT26 cells, cDNA was prepared, and the genes for the dormancy markers NR2F1 and p21 or the EMT-TFs Slug and Zeb1 were amplified by qPCR as described in the methods. (A–D) cells incubated in co-cultures (n = 5–6), (E–H) single cultures incubated with the addition of TGFβ (10 ng/mL) (n = 5–6), and (I–L) cells incubated with the addition of recombinant EMMPRIN (25 and 250 ng/mL) (n = 5). Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Expressing, Co-Culture Assay, Incubation, Cell Culture, Recombinant

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 5. CT26-KD exhibits reduced proliferation. CT26-WT or CT26-KD cells (8 × 104 cells) were incubated under the same conditions as described in Figure 4. Cell proliferation was measured using (A,D,G) the CCK8 kit (n = 8) or (H) the BrdU kit (n = 7) as well as the expression of (B,E,I) the cyclin D1 mRNA (n = 5) or (C,F), and the Ki67 mRNA (n = 4). (A–C) Cells incubated in co-cultures, (D,F) single cultures incubated with the addition of TGFβ (10 ng/mL), and (G–I) cells incubated with the addition of recombinant EMMPRIN (25 and 250 ng/mL). Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test. ***, p < 0.001 relative to the CT26-WT without addition of rec. EMMPRIN; $$$, p < 0.001 relative to CT26-WT with 25 ng/ml rec. EMMPRIN; &&&, p < 0.001 relative to the CT26-WT at each concentration.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Incubation, Expressing, Recombinant, Concentration Assay

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 6. CT26-KD exhibits reduced angiogenic potential, and the co-culture reverses it. The mouse endothelial cell line bEND3 (4 × 104 cells) was cultured in full medium in 96-well plates to confluency for 24 h. A scratch was made across the monolayer, detached cells were washed away, and the remaining bEND3 cells were incubated with conditioned media (CM) derived from previous experiments for 24 h, in order to allow the migration of cells to close the gap. The CM was diluted 1:2 with full medium, to a final volume of 100 µL. Images were taken before the addition of the CM (0 h) and after 24 h of incubation with the CM (24 h). (A) Representative images of the wound assay of co-cultures. Bar size is 250 µM. (B) Quantitation of the migration of bEND3 cells cultured with CM from co-culture experiments (n = 12), with (C) concentrations of VEGF (n = 7) and (D) MMP-9 (n = 7) in the supernatants derived from co-culture experiments. (E) Quantitation of the migration using CM derived from the TGFβ experiments (n = 5), and concentrations of (F) VEGF (n = 8) and (G) MMP-9 (n = 5) in the supernatants derived from TGFβ experiments. (H) Quantitation of the migration using CM derived from the recombinant EMMPRIN experiments (n = 9), and concentrations of (I) VEGF (n = 9) and (J) MMP-9 (n = 7) in the supernatants derived from recombinant EMMPRIN experiments. Data are presented as means ± SE, and analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Co-Culture Assay, Cell Culture, Incubation, Derivative Assay, Migration, Quantitation Assay, Recombinant

Journal: Biomedicines

Article Title: Knocking-Down CD147/EMMPRIN Expression in CT26 Colon Carcinoma Forces the Cells into Cellular and Angiogenic Dormancy That Can Be Reversed by Interactions with Macrophages.

doi: 10.3390/biomedicines11030768

Figure Lengend Snippet: Figure 7. The combination of recombinant TGFβ and EMMPRIN has no effect on the addition of TGFβ or EMMPRIN alone. CT26-WT or CT26-KD cells (2.5 × 104 cells) were incubated in triplicates for 48 h in serum starvation medium, with or without the addition of recombinant TGFβ (5 ng/mL), EMMPRIN (5 ng/mL), or their combination. The effect on the (A) proliferation, as measured by CCK8 (n = 10), (B) the concentrations of secreted VEGF (n = 5), as well as the mRNA expression of (C) NR2F1 (n = 6), (D) p21 (n = 6), (E) Slug (n = 6), and (F) Zeb1 (n = 6). Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: Membranes were blocked with the block-Chemi buffer (Advansta) overnight at 4 ◦C, and then incubated with the primary antibody (goat anti-mouse EMMPRIN, R&D systems) diluted 1:1000 for 1 h at room temperature.

Techniques: Recombinant, Incubation, Expressing

Journal: bioRxiv

Article Title: EMMPRIN confers metabolic advantage for monocytes and macrophages to promote disease in a model of multiple sclerosis

doi: 10.1101/2024.08.11.607460

Figure Lengend Snippet: A) Confocal images of perivascular cuffs in the cerebellar white matter of CCR2:EMMP +/+ and CCR2:EMMP −/− D18 EAE showing pan-leukocytic marker, CD45 (red) encased in basement membrane delineated by laminin staining (green). Insets showing enlarged cuffs with CD45+ cells within. Scale-50μm. B) Histograms denoting average number of cuffs observed in the two groups. Data analyzed by student’s T-test. **p<0.05, N of 6 per group. C) Spinal cords were harvested from CCR2:EMMP +/+ and CCR2:EMMP −/− mice at D18 after immunization and subjected to flow cytometry: Singlet viable cells were gated on CD11b and CD45 for CD45hi CD11b+ monocyte/macrophages as shown in dot-plot for CCR2:EMMP −/− and CCR2:EMMP +/+ examples; there were few monocyte/macrophages in the spinal cord of CCR2:EMMP −/− mice. D) Analysis of % EMMPRIN+ CCR2+ infiltrated macrophages as well as E) expression of EMMPRIN levels (MFI = mean fluorescence intensity) in CCR2+ CD11b+LY6G-cells. F) Dot plot exhibiting Ly6G and CD11b staining from blood of CCR2:EMMP +/+ and CCR2:EMMP −/− D12 EAE mice which is quantified as %CD11b+ Ly6G-CD45+ cells in (G) . Histograms showing %CD11b+ Ly6G-CD45+ cells from D18 EAE in WT, CCR2:EMMP +/+ and CCR2:EMMP −/− mice. Flow plots shown in D, E, G and H were compared using one-way ANOVA with Bonferroni post-hoc test. *p<0.05, **p<0.01 and ***p<0.001. Data represented as mean ± SD.

Article Snippet: Cells were then washed and incubated in following antibodies for 30 minutes at 4°C in the dark: PerCP rat anti-mouse CD45 (557235, BD Biosciences, 1:50), FITC rat anti-CD11b (553310, BD Biosciences, 1:50), APC-Cy7 rat anti-mouse Ly6G (560600, BD Biosciences, 1:50), PE rat anti-mouse EMMPRIN (562676, BD Biosciences, 1:50), BUV395 rat anti-mouse CD115 (564059, BD Biosciences, 1:50), BV510 rat anti-mouse CCR2 (CD192, 747970, BD Biosciences, 1:50) and fixable viability dye eFluor 780 (65-0865-14, eBioscience).

Techniques: Marker, Membrane, Staining, Flow Cytometry, Expressing, Fluorescence

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