Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 1. Cardiac fibrosis and innervation. (A) Masson’s trichrome staining of young (3-month-old) and old (10-month-old) WT and mdx mouse hearts. Scale bars, 100 μm. The graph shows the area as a percentage of the fibrosis index (fibrotic area/whole area). N = 3 biological replicates. (B) qPCR for fibrosis-related transcripts in 3- and 10-month-old mdx and WT hearts (Col1a1, Col3a1, Fn1, Tgfb1, Twist 1, Twist 2). N = 3 biological replicates. (C) Confocal images for tyrosine hydroxylase (TH), Synapsin 1 (SYN1), MPs (F4/80), proteoglycan CSPG4, and fibronectin 1 (FN1) on myocardium sections of 3- and 10-month-old mice. The markers TH, SYN1, and CSPG4 are labeled in yellow, cardiac troponin (cTNNT) and F4/80 are in magenta, while FN1 is in white. Scale bars, 10 μm. Nuclei are counterstained with DAPI in blue. (D) Charts indicate positive area expressed as percentage of TH, SYN1, and F4/80 on whole area. N = 3 sections per N = 3 biological replicates. (E and F) Western blot analysis of TH, SYN1, and CSPG4 in mdx and WT hearts of young and old mice. Graphs show optical density (OD) of protein bands normalized to vinculin (VCL). N = 3 biological replicates. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were calculated using Student’s t-test.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Staining, Labeling, Western Blot

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 2. MPs as main producers of CSPG4. (A) Graphic illustration of CSPG4-CAR-Ts cells against MPs expressing CSPG4. Created with Biorender.com. Diagrams quantifying the percentage of live cells of bone marrow-derived MPs of both WT and mdx M0 (nonactivated), M1 (pro-inflammatory) and M2 (anti-inflammatory), or CFs after exposure to CSPG4.CARTs compared to cells co-cultured with control CART (cCART). N = 3 biological replicates. (B) ELISA assay for CSPG4 in supernatants (SUP) isolated from WT and mdx MPs. The chart illustrates the optical density (OD) of CSPG4 in the two groups. Each dot represents the eluted fractions, N = 16. (C) FACSymphony of cardiac tissues. The first chart represents a scatter plot of t-SNE relative to concatenated total cells from 3- or 10-month-old WT and mdx hearts. The second chart illustrates the distribution of CD45neg cells (light pink cluster) and myeloid cells (dark pink cluster) in all groups. (D) Graph of FACS analysis relative to number of total MPs (F4/80) positive for CSPG4 in 3- or 10-month-old WT and mdx hearts. (E) Expression of CSPG4 in subpopulations of MPs: CD80pos (M1), CD206 pos (M2), Ly6Clow (resident), and Ly6Chigh (monocytes) in 3- or 10-month-old WT and mdx hearts. Error bars show SEM. *p < 0.05, **p < 0.01 calculated using Student’s t-test and one-way ANOVA.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Expressing, Derivative Assay, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Isolation

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 3. MPs release CSPG4 in media and inhibit axon growth of dorsal root ganglia. (A) Rendering of 3D NCJ generation. The picture was created with Biorender.com and illustrates the composition of the 3D NCJ model: WT dorsal root ganglia (DRG), WT cardiomyocytes (CMs), and WT CFs were encapsulated in PEG-FB hydrogel conditioned with the supernatants derived from MP cultures of WT, WT + CSPG4 blocking antibody (BAb), mdx, and mdx + CSPG4 (BAb). (B) Brightfield images of CFs, CMs, and DRG after 1 day of culture (upper panel) and 3D constructs of NCJ models after 7 days (lower panel). Scale bars, 100 μm. (C) Confocal images of 3D NCJ models: WT, WT + CSPG4 BAb, mdx, mdx + CSPG4 BAb. DRG are labeled with tubulin beta 3 (TUBB3, in yellow), CMs with cardiac troponin (cTNNT, in magenta), and CFs with fibroblast marker (ER- TR7, in white). (D) The graph indicates the area (%) occupied by neuron endings labeled by TUBB3 inside the 3D NCJ models. N = 3 biological replicates, N = 2 sections/constructs. Scale bars, 10 μm. **p < 0.01, ***p < 0.001 were determined using one-way ANOVA.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Derivative Assay, Blocking Assay, Construct, Labeling, Marker

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 4. Cardiac innervation in old mdx and WT mice. (A) Immunofluorescence staining for tyrosine hydroxylase (TH), Synapsin 1 (SYN1), total MPs (F4/80) and proteoglycan (CSPG4), fibronectin 1 (FN1) on cardiac sections from 10-month-old mice. The markers TH and SYN1 are labeled in yellow, while cardiac troponin (cTNNT) and F4/80 are in magenta, FN1 in white, and CSPG4 in yellow or white. DAPI nuclear counterstaining is in blue. Scale bars, 10 μm. (B) Charts indicate area expressed as percentage of TH, SYN1, and F4/80 (positive area/whole area). N = 3 sections per N = 3 biological replicates. (C and D) Western blot analysis for TH, SYN1, and CSPG4 quantified as optical density (OD) of protein bands normalized to vinculin (VCL). N = 3 biological replicates. (E) qPCR relative to markers for neurotransmitter production (Th), survival (Ngf, Ntrk1, and Ngfr) and axon guidance (Tubb3) in DRG. N = 3 biological replicates. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were calculated using one-way ANOVA.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Staining, Labeling, Western Blot

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 5. Histological analysis and gene profile of cardiac tissue after treatment with givinostat in old mdx and WT mice. (A) Confocal images of immunostaining for gap junctions (CX43) and cardiomyocytes positive for cardiac troponin (cTNNT), N = 3 sections per N = 3 biological replicates. Scale bars, 50 μm. (B) Charts indicate area as percentage of CX43 (positive area/whole area). (C and D) Representative Masson’s trichrome images of WT, mdx saline, and mdx Giv hearts and fibrosis index quantification (fibrotic area/whole area) after 60 days of treatment. Scale bars, 100 μm. N = 3 biological replicates. (E) qPCR of fibrosis-related genes (Tgfb1, Col1a1, Col3a1, Twist1, Twist2, Fn1), cell surface proteoglycan (Cspg4); the enzyme catalyzes the transfer of sulfate groups on CSPG4 chains (Chst11) and inflammatory markers (Adgre1 and Mmp9). N = 3 biological replicates. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were calculated using one-way ANOVA and Student’s t-test.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Immunostaining, Saline

Journal: PloS one

Article Title: Pro-inflammatory cytokines reduce the proliferation of NG2 cells and increase shedding of NG2 in vivo and in vitro.

doi: 10.1371/journal.pone.0109387

Figure Lengend Snippet: Figure 2. Proliferation of NG2+ cells in response to systemic lipopolysaccharide treatment. Unaltered number of NG2+/ki67+ cell numbers was found in the medial prefrontal cortex (mPFC) (A), the basolateral nucleus (BL) (B), the dentate hilus (Hilus) (C) and the molecular layer (ML) (D) 2 h after lipopolysaccharides (LPS) treatment compared to saline treatment (Saline). In contrast, a significant decreased number of NG2+/ ki67+ cells were detected in the BL (B), the Hilus (C) and the ML (D), but not in the mPFC (A), 24 h after LPS treatment compared to saline treatment. The number of Ki67+ cell in the subgranular zone (SGZ) of the hippocampal granular cell layer (E) was also unaltered after 2 h, but significantly reduced 24 h after LPS compared to saline treatment. *** indicates a significant difference at p,0.001 level. ** indicates a significant difference at p, 0.01 level. * indicates a significant difference at p,0.05 level. Images in (F–H) demonstrate a double immunoflourescence staining with antibodies directed against the endogenous proliferation marker Ki67 (F) and NG2 (G). Images (F) and (G) are superimposed in (H). Scale bar = 10 mm. doi:10.1371/journal.pone.0109387.g002

Article Snippet: Recombinant rat NG2 (kind gift from Dr William Stallcup) or recombinant human NG2 (Origene technologies) was used as standard.

Techniques: Saline, Staining, Marker

Journal: PloS one

Article Title: Pro-inflammatory cytokines reduce the proliferation of NG2 cells and increase shedding of NG2 in vivo and in vitro.

doi: 10.1371/journal.pone.0109387

Figure Lengend Snippet: Figure 4. Proliferation and supernatant sNG2 levels of cultured human NG2 cells after exposure cytokines. The left bar graph shown the significantly increased sNG2 levels found in cell culture supernatant after 24 h stimulation with interleukin (IL)-1b (0.3 ng/ml), IL-6 (1.5 ng/ml) and IL-4 (150 pg/ml). The sNG2 levels were not altered in response to 20 ng/ml IL-8. The right bar graph demonstrates the significantly decreased EdU + cell area to DAPI cell area ratio after exposure to IL-1b and IL-6, but not after IL-8 or IL-4. * indicates a significant difference at p,0.05 level, ** indicates a significant difference at p,0.01 level. doi:10.1371/journal.pone.0109387.g004

Article Snippet: Recombinant rat NG2 (kind gift from Dr William Stallcup) or recombinant human NG2 (Origene technologies) was used as standard.

Techniques: Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants

doi: 10.1074/jbc.M117.806687

Figure Lengend Snippet: Characterization of defective purified TcdB mutants Y1824K and N1839K. A, pore formation of purified mutant toxins was tested on CHO cells preloaded with 86Rb+. Pore formation was induced by acidification of the external medium (black bars, control, pH 7.5; gray bars, pH 4.5) n = 4. B, relative gene expression of TcdB receptors. The relative gene expression of identified TcdB receptors NECTIN3, CSPG4, and FZDs on different mammalian cell lines was assessed using a ΔΔCq method determined from quantitative PCR data (31) using β-actin, ACTB, as the reference gene (n = 2). Vero cells that were used in the cell viability assay are highlighted in the red box. C, cell-surface binding of defective mutant toxins to target cells. Shown is an immunoblot analysis of TcdB wild type and mutants bound to the Vero cell surface at 4 °C. The cells were exposed to 200 ng/ml TcdB for 30 min before being lysed for immunoblot analysis. Membrane-bound proteins were detected by anti-TcdB antibody and rabbit anti-cadherin antibody as loading control.

Article Snippet: pCMV6-CSPG4 vector expressing full-length Myc-DDK-tagged CSPG4 was purchased from OriGene Technologies (catalog no. RC218462).

Techniques: Purification, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Viability Assay, Binding Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants

doi: 10.1074/jbc.M117.806687

Figure Lengend Snippet: Characterization of receptor binding of defective mutants. A, association of ectodomain of NECTIN3 with TcdB wild type and mutants, assayed by co-immunoprecipitation (CO-IP) analysis. His-tagged TcdB (3 μg) was incubated with 5 μg of the His-tagged NECTIN3 ectodomain at 4 °C for 16 h. TcdB and NECTIN3 interaction was immunoprecipitated and detected using anti-His antibody. B, interaction of Fzd7 with TcdB by gel filtration assay. Purified Fzd7-CRD mVenus was mixed with TcdB at 1:5 molar ratios of TcdB:Fzd7 and incubated at room temperature for 30 min and run on a Superose 6 Increase 10/300 GL column. Elution peaks of only TcdB (black) and the complex with both TcdB and Fzd7 (red) were monitored by absorbance at 280 nm, and the complex was visualized on SDS-PAGE. Note that all samples were run on the same gel; however, to simplify the figure, the relevant portion of the gel was shown with the corresponding column elution profile. Accordingly, the molecular weight marker was reused in all panels. C, binding of CSPG4 to TcdB wild type and mutants, assayed by co-immunoprecipitation analysis. TcdB (3 μg) was mixed incubated with 150 μg of cell lysate from HEK293 cells overexpressing FLAG-tagged CSPG4 at 4 °C for 2 h. The complex was immunoprecipitated using anti-FLAG magnetic beads and visualized by anti-FLAG (CSPG4) and anti-His (TcdB) antibodies. D, immunoblot analysis of the wild type (CSPG4+/+) and CSPG4 knock-out (CSPG4−/−) Vero cell lysates for expression of CSPG4 and GAPDH (control). E, sensitivity of wild type (CSPG4+/+) and CSPG4−/− Vero cells toward TcdB wild type and mutants. Indicated Vero cells were exposed to TcdB (wild type, Y1824K, and N1839K) and subjected to cell survival using the SRB assay. F, toxicity of TcdA wild type and mutants (Y1822K and N1837K) on CHO cells, measured by Prestoblue fluorescence assay as described.

Article Snippet: pCMV6-CSPG4 vector expressing full-length Myc-DDK-tagged CSPG4 was purchased from OriGene Technologies (catalog no. RC218462).

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Filtration, Purification, SDS Page, Molecular Weight, Marker, Magnetic Beads, Western Blot, Knock-Out, Expressing, Sulforhodamine B Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants

doi: 10.1074/jbc.M117.806687

Figure Lengend Snippet: Characterization of TcdB C-terminal truncations. A, schematic drawings of TcdB C-terminal truncated constructs, B2034 (TcdB1–2034), B1900 (TcdB1–1900), B1834 (TcdB1–1834), and CROP domain (TcdB1834–2366). B, interaction between truncated TcdB constructs and CSPG4. His-tagged TcdB constructs were incubated with lysate from cells overexpressing FLAG-tagged CSPG4 and subjected to immunoprecipitation (CO-IP) by anti-FLAG antibody and visualized by immunoblot as described. C, sensitivity of Vero and Caco-2 cells toward TcdB C-terminal truncations. Vero cells (left) and Caco-2 cells (right) were exposed to TcdB-truncated toxins, and cell survival was assessed by the SRB assay as previously described.

Article Snippet: pCMV6-CSPG4 vector expressing full-length Myc-DDK-tagged CSPG4 was purchased from OriGene Technologies (catalog no. RC218462).

Techniques: Construct, Incubation, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Sulforhodamine B Assay

Journal: The Journal of Biological Chemistry

Article Title: Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants

doi: 10.1074/jbc.M117.806687

Figure Lengend Snippet: Binding model of CSPG4 and bezlotoxumab to TcdB. A, a homology model of the TcdB segment 1800–1833 with downstream CROP domain was built based on sequence and structural alignment via Phyre 2.0 (yellow) and the crystal structure of CROPs (green) with two Fab domains of bezlotoxumab from Orth et al. (27). B, model of structure of TcdB segment 1800–1833 and CROP domain with bezlotoxumab-binding sites, E1 and E2, and identified key residues mapped to the structure. C, inhibition of CSPG4 binding to TcdB by bezlotoxumab and actoxumab (control) by immunoprecipitation (CO-IP) assay as described previously.

Article Snippet: pCMV6-CSPG4 vector expressing full-length Myc-DDK-tagged CSPG4 was purchased from OriGene Technologies (catalog no. RC218462).

Techniques: Binding Assay, Sequencing, Inhibition, Immunoprecipitation, Co-Immunoprecipitation Assay