Journal: Molecular and Cellular Biology

Article Title: CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum

doi: 10.1128/mcb.00383-18

Figure Lengend Snippet: Figure 1. Mutations in CD19 exon 2 result in cell surface localization defects. A) Schematic of VSVg-CD19 FL, VSVg-105R and VSVg-Δ2 retroviral constructs. The VSVg tag was inserted in exon 1 right after the signal peptide sequence to prevent cleavage of the epitope. B) Western blotting with anti-VSVg antibody in transduced Nalm6 cell lines. C) Flow cytometry of live or fixed/permeabilized transduced cell lines stained with anti-VSVg-FITC or anti- CD19 (FMC63)-PE antibody. D) Flow cytometry of live transduced Nalm6 cell lines grown at 37°C or 32°C for 72 hours before staining with anti-VSVg-FITC for 1 hour. Cells were then washed with PBS as a control or Glycine to dissociate any surface bound antibody. E-F) Killing assays using anti-VSVg antibody and anti-mouse-MMAF secondary antibody drug conjugate (2°ADC) in transduced Nalm6 cell lines grown at 37°C (E) or 32°C (F) for 72 hours.

Article Snippet: 413 Antibodies 414 The following antibodies were used for protein detection: α-CD19-PE (Beckman Coulter #IM1285U), α-415 CD19 (Cell Signaling #3574), α-CD19 3G7, (Origen #TA506234), α-CD19 HD37 (Thermo Fisher #MA1-416 34005), α-VSVg-FITC (Abcam #ab3863), α-VSVg (Abcam # ab50549), α-CD81 (LifeSpan #LS-417 C359239), α-CD81-FITC (Molecular Probes #A15753), α-calnexin (Cell Signaling #2679), α-calreticulin 418 on A ugust 15, 2018 by guest http://m cb.asm .org/ D ow nloaded from (Cell Signaling #12238), α-UGCGL1 (NovoPro Labs # 116554), α-PDI (Cell Signaling #3501), α-Mouse 419 Alexa 488 (Thermo Fisher #R37114), α-rabbit Alexa 594 (Thermo Fisher #A-21207).

Techniques: Retroviral, Construct, Sequencing, Western Blot, Flow Cytometry, Staining, Control

Journal: Molecular and Cellular Biology

Article Title: CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum

doi: 10.1128/mcb.00383-18

Figure Lengend Snippet: Figure 2. CD19ex2 variants exhibit altered glycosylation patterns. A) Schematic showing cellular compartments where sequential steps of protein glycosylation and processing occur. B) Schematic showing the intended targets of cleavage by EndoH and EndoF glycosidases as well as the α-mannosidase inhibitor Swainsonine. C) Western blot with anti-CD19 or anti-actin antibody of protein extracts from indicated transduced Nalm6 cell lines after treatment with DMSO or 10µg/ml Swainsonine for 24hr. D) Western blot with anti-CD19 or anti-actin antibody of lysates from indicated transduced 697 cell lines following treatment with DMSO or 10µg/ml Swainsonine for 24hr. E) Western blot with anti-CD19 or anti-actin antibody of protein lysates from indicated transduced Nalm6 cell lines following in vitro mock or PNGase F treatment. F) Western blot with anti-CD19, or anti-actin antibody of protein lysates from indicated transduced 697 cell lines following in vitro mock or PNGase F treatment. G) Nalm6-ΔCD19 cells or cells transduced with CD19-FL or CD19ex2var constructs were radiolabeled for 15 min, chased for 1 or 2 h, and immunoprecipitated using a monoclonal antibody against human CD19. Immunoprecipitates were treated with endo-H or PNGase F before analysis on an SDS-PAGE gel. CHO, high mannose-type glycans; CHO*, complex-type glycans; NAG, N-acetylglucosamine.

Article Snippet: 413 Antibodies 414 The following antibodies were used for protein detection: α-CD19-PE (Beckman Coulter #IM1285U), α-415 CD19 (Cell Signaling #3574), α-CD19 3G7, (Origen #TA506234), α-CD19 HD37 (Thermo Fisher #MA1-416 34005), α-VSVg-FITC (Abcam #ab3863), α-VSVg (Abcam # ab50549), α-CD81 (LifeSpan #LS-417 C359239), α-CD81-FITC (Molecular Probes #A15753), α-calnexin (Cell Signaling #2679), α-calreticulin 418 on A ugust 15, 2018 by guest http://m cb.asm .org/ D ow nloaded from (Cell Signaling #12238), α-UGCGL1 (NovoPro Labs # 116554), α-PDI (Cell Signaling #3501), α-Mouse 419 Alexa 488 (Thermo Fisher #R37114), α-rabbit Alexa 594 (Thermo Fisher #A-21207).

Techniques: Glycoproteomics, Western Blot, In Vitro, Transduction, Construct, Immunoprecipitation, SDS Page

Journal: Molecular and Cellular Biology

Article Title: CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum

doi: 10.1128/mcb.00383-18

Figure Lengend Snippet: Figure 3. Inhibition of CD19 glycosylation does not affect its subcellular localization. A) Immuno- fluorescence of Nalm6-ΔCD19 cells expressing FL-CD19-GFP (top schematic) after treatment with DMSO or Swainsonine for 24hr. Plasma membrane is stained with wheat germ agglutinin-Alexa 647 (red) and nuclei are stained with DAPI (blue). Histogram showing colocalization of CD19-GFP (green) and plasma membrane (red) channels. B) Pearson’s correlation colocalization analyses for FL-CD19-GFP and plasma membrane. Three separate fields containing at least 100 cells were analyzed for each condition. C) Immunofluorescence of 697-ΔCD19 cells expressing CD19-FL- GFP after treatment with DMSO or Swainsonine for 24hr. Plasma membrane is stained by wheat germ agglutinin-Alexa 647(red) and nuclei are stained with DAPI (blue). Histogram showing colocalization of CD19-GFP (green) and plasma membrane (red) channels. D) Pearson’s correlation colocalization analyses for FL-CD19-GFP and plasma membrane. Three separate fields containing at least 100 cells were analyzed for each condition. E) Immunofluorescence of Nalm6- ΔCD19 cells transduced with N86A-CD19-GFP fusion protein (top schematic). Plasma membrane is stained with wheat germ agglutinin-Alexa 647 (red).

Article Snippet: 413 Antibodies 414 The following antibodies were used for protein detection: α-CD19-PE (Beckman Coulter #IM1285U), α-415 CD19 (Cell Signaling #3574), α-CD19 3G7, (Origen #TA506234), α-CD19 HD37 (Thermo Fisher #MA1-416 34005), α-VSVg-FITC (Abcam #ab3863), α-VSVg (Abcam # ab50549), α-CD81 (LifeSpan #LS-417 C359239), α-CD81-FITC (Molecular Probes #A15753), α-calnexin (Cell Signaling #2679), α-calreticulin 418 on A ugust 15, 2018 by guest http://m cb.asm .org/ D ow nloaded from (Cell Signaling #12238), α-UGCGL1 (NovoPro Labs # 116554), α-PDI (Cell Signaling #3501), α-Mouse 419 Alexa 488 (Thermo Fisher #R37114), α-rabbit Alexa 594 (Thermo Fisher #A-21207).

Techniques: Inhibition, Glycoproteomics, Fluorescence, Expressing, Clinical Proteomics, Membrane, Staining, Immunofluorescence, Transduction

Journal: Molecular and Cellular Biology

Article Title: CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum

doi: 10.1128/mcb.00383-18

Figure Lengend Snippet: Figure 4. CD19ex2 variants localize in the endoplasmic reticulum. A) Schematic of the CD19-FL, CD19Δex2 or CD19-105R-GFP fusion protein constructs expressed in Nalm6-ΔCD19 cells. B) Western blot with anti-CD19 antibody of lysates from indicated cell lines. C) Live cell flow cytometry for Empty (black), CD19-FL (blue), CD19-Δex2 (green) and CD19-105R (red) using anti-CD19-PE antibody. D) Immunofluorescence confocal microscopy of GFP- construct transduced cell lines (green). Plasma membrane was stained with wheat germ agglutinin-Alexa 647 (red). Endoplasmic reticulum was stained with anti-calnexin (Cell Signaling)/anti-Rabbit-594 (cyan). Histogram localization analysis showing overlaps of channels. E) Pearson’s correlation coefficient analyses shows strong colocalization of plasma membrane and CD19-FL and endoplasmic reticulum and CD19ex2var isoforms.

Article Snippet: 413 Antibodies 414 The following antibodies were used for protein detection: α-CD19-PE (Beckman Coulter #IM1285U), α-415 CD19 (Cell Signaling #3574), α-CD19 3G7, (Origen #TA506234), α-CD19 HD37 (Thermo Fisher #MA1-416 34005), α-VSVg-FITC (Abcam #ab3863), α-VSVg (Abcam # ab50549), α-CD81 (LifeSpan #LS-417 C359239), α-CD81-FITC (Molecular Probes #A15753), α-calnexin (Cell Signaling #2679), α-calreticulin 418 on A ugust 15, 2018 by guest http://m cb.asm .org/ D ow nloaded from (Cell Signaling #12238), α-UGCGL1 (NovoPro Labs # 116554), α-PDI (Cell Signaling #3501), α-Mouse 419 Alexa 488 (Thermo Fisher #R37114), α-rabbit Alexa 594 (Thermo Fisher #A-21207).

Techniques: Construct, Western Blot, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Clinical Proteomics, Membrane, Staining

Journal: Molecular and Cellular Biology

Article Title: CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum

doi: 10.1128/mcb.00383-18

Figure Lengend Snippet: Figure 5. Disruption of CD19 Cys38-Cys97 disulfide bond leads to loss of surface expression. A) Schematic representation of the CD19 protein indicating the exon2 residues involved in bisulfite bonds and loop formation, and glycosylation. Depictions of the C97A and N86A/C97A-CD19 protein constructs are also shown. B) I- Tasser protein structure prediction of CD19-FL (top panel) and CD19-105R (bottom panel). For simplicity, only exons 1 and 2 are shown. C) Western blot with anti-CD19 or anti-actin antibody of protein extracts from indicated transduced Nalm6 cell lines following treatment with DMSO or 10ug/mL Swainsonine for 24 hours. Various degrees of CD19 protein glycosylation are shown by gel-shift of bands. CHO, high mannose-type glycans; CHO*, complex-type glycans; NAG, N-acetylglucosamine. D) Western blot with anti-CD19 or anti-actin antibody of protein lysates from indicated transduced Nalm6 cell lines following in vitro mock or PNGase F treatment. E) Western blot with anti- CD19, or anti-actin antibody of protein lysates from indicated transduced 697 cell lines following in vitro mock or PNGase F treatment. F) Live cell flow cytometry using α-CD19-PE antibody of transduced Nalm6 and 697 cell lines.

Article Snippet: 413 Antibodies 414 The following antibodies were used for protein detection: α-CD19-PE (Beckman Coulter #IM1285U), α-415 CD19 (Cell Signaling #3574), α-CD19 3G7, (Origen #TA506234), α-CD19 HD37 (Thermo Fisher #MA1-416 34005), α-VSVg-FITC (Abcam #ab3863), α-VSVg (Abcam # ab50549), α-CD81 (LifeSpan #LS-417 C359239), α-CD81-FITC (Molecular Probes #A15753), α-calnexin (Cell Signaling #2679), α-calreticulin 418 on A ugust 15, 2018 by guest http://m cb.asm .org/ D ow nloaded from (Cell Signaling #12238), α-UGCGL1 (NovoPro Labs # 116554), α-PDI (Cell Signaling #3501), α-Mouse 419 Alexa 488 (Thermo Fisher #R37114), α-rabbit Alexa 594 (Thermo Fisher #A-21207).

Techniques: Disruption, Expressing, Glycoproteomics, Construct, Western Blot, Gel Shift, In Vitro, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum

doi: 10.1128/mcb.00383-18

Figure Lengend Snippet: Figure 6. Disruption of CD19 Cys38-Cys97 disulfide bond leads to endoplasmic reticulum retention. A) Immuno-fluorescence confocal microscopy of indicated CD19-GFP-construct (green) transduced Nalm6 cell lines. Plasma membrane is stained with wheat germ agglutinin-Alexa 647 (red), endoplasmic reticulum is stained with anti- calnexin (Cell Signaling)/anti-Rabbit-594 antibody (cyan) and nuclei are stained with DAPI (blue). Histogram localization analysis shows overlap of CD19-GFP, ER/Calnexin and plasma membrane channels. B) Pearson’s correlation colocalization analyses of green (CD19) and red (plasma membrane) channels or green (CD19) and ER/calnexin channels for indicated Nalm6 cell lines. Three separate fields containing at least 100 cells were analyzed for each condition. C) Immunofluorescence confocal microscopy of indicated CD19-GFP-construct (green) transduced 697 cells. Plasma membrane is stained with wheat germ agglutinin-Alexa 647 (red) and nuclei are stained with DAPI (blue). Histogram localization analysis shows overlap of CD19-GFP (green) and plasma membrane (red). D) Pearson’s correlation colocalization analyses of green (CD19) and red (plasma membrane) channels for indicated 697 cell lines. Three separate fields containing at least 100 cells were analyzed for each condition.

Article Snippet: 413 Antibodies 414 The following antibodies were used for protein detection: α-CD19-PE (Beckman Coulter #IM1285U), α-415 CD19 (Cell Signaling #3574), α-CD19 3G7, (Origen #TA506234), α-CD19 HD37 (Thermo Fisher #MA1-416 34005), α-VSVg-FITC (Abcam #ab3863), α-VSVg (Abcam # ab50549), α-CD81 (LifeSpan #LS-417 C359239), α-CD81-FITC (Molecular Probes #A15753), α-calnexin (Cell Signaling #2679), α-calreticulin 418 on A ugust 15, 2018 by guest http://m cb.asm .org/ D ow nloaded from (Cell Signaling #12238), α-UGCGL1 (NovoPro Labs # 116554), α-PDI (Cell Signaling #3501), α-Mouse 419 Alexa 488 (Thermo Fisher #R37114), α-rabbit Alexa 594 (Thermo Fisher #A-21207).

Techniques: Disruption, Fluorescence, Confocal Microscopy, Construct, Clinical Proteomics, Membrane, Staining, Immunofluorescence

Journal: Molecular and Cellular Biology

Article Title: CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum

doi: 10.1128/mcb.00383-18

Figure Lengend Snippet: Figure 7. Endogenous CD19ex2 variants generated by genome editing are also retained in the endoplasmic reticulum. A) Schematic of CRISPR/Cas9 mutant clones to model CD19-Δex2 (clone 11 ) or CD19- 105R (clone 12). Western blot with anti-CD19 or anti-Gapdh antibodies to compare protein expression between CRISPR/Cas9 and retroviral overexpression cell line models. B) Flow cytometry of live or fixed and permeabilized Nalm6 Parental and CRISPR/Cas9 modified cells expressing endogenous CD19 Wild type (parental) , CD19-Δex2 (clone 11) and CD19-105R-like (clone 12) isoforms. C) Killing assay using anti-CD19 antibody and anti-mouse-DMDM secondary antibody drug conjugate (2°ADC) in Nalm6 parental (CD19-WT), ΔCD19 (null), clone 11 (CD19-Δex2) or clone 12 (CD19-105R-like). D) Nalm6-ΔCD19, parental, clone 11 or clone 12 cell lines were radiolabeled for 15 min, chased for 1 or 2 h, and immunoprecipitated using a monoclonal antibody against human CD19. Immuno- precipitates were treated with endo-H or PNGase F before analysis on an SDS-PAGE gel. CHO, high mannose-type glycans; CHO*, complex-type glycans; NAG, N-acetylglucosamine. E) Immunoblotting with anti-CD19 or anti- calnexin antibodies following immunoprecipitation with anti-CD19 antibody in indicated cell lines. F) Immunofluorescence confocal microscopy of Nalm6 Parental (CD19-WT), clone 11 (CD19-Δex2) or clone 12 (CD19- 105R-like). Cells are stained with anti-CD19(3G7 OriGene )/anti-Mouse-488 (green). Plasma membrane is stained with Wheat germ agglutinin-Alexa 647 (red), endoplasmic reticulum is stained with anti-calnexin (Cell Signaling)/anti-Rabbit-594 antibody (cyan) and nuclei are stained with DAPI (blue). Histogram localization analysis shows overlap of color channels.

Article Snippet: 413 Antibodies 414 The following antibodies were used for protein detection: α-CD19-PE (Beckman Coulter #IM1285U), α-415 CD19 (Cell Signaling #3574), α-CD19 3G7, (Origen #TA506234), α-CD19 HD37 (Thermo Fisher #MA1-416 34005), α-VSVg-FITC (Abcam #ab3863), α-VSVg (Abcam # ab50549), α-CD81 (LifeSpan #LS-417 C359239), α-CD81-FITC (Molecular Probes #A15753), α-calnexin (Cell Signaling #2679), α-calreticulin 418 on A ugust 15, 2018 by guest http://m cb.asm .org/ D ow nloaded from (Cell Signaling #12238), α-UGCGL1 (NovoPro Labs # 116554), α-PDI (Cell Signaling #3501), α-Mouse 419 Alexa 488 (Thermo Fisher #R37114), α-rabbit Alexa 594 (Thermo Fisher #A-21207).

Techniques: Generated, CRISPR, Mutagenesis, Clone Assay, Western Blot, Expressing, Retroviral, Over Expression, Flow Cytometry, Modification, Immunoprecipitation, SDS Page, Immunofluorescence, Confocal Microscopy, Staining, Clinical Proteomics, Membrane

Journal: Molecular and Cellular Biology

Article Title: CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum

doi: 10.1128/mcb.00383-18

Figure Lengend Snippet: Figure 8. CD19ex2 variants preferentially interact with ER/MHC-related proteins. A) Immunoprecipitation of CD19 in lysates from indicated transduced Nalm6 cell lines were subjected to mass spectrometry. The bars represent normalized total spectra for the corresponding identified protein targets. B) Heat map showing proteins from the Reactome Adaptive Immune System gene-set that showed differential binding to CD19, CD19-105R and CD19-Δex2, as determined by mass spectrometry. C) Double stained live cell flow cytometry using anti-CD19-PE and anti-CD81-FITC in Nalm6 parental and Nalm6-ΔCD81 cells lines. D) Immunoblotting of CD81, calnexin, Calreticulin, UGCGL1 and PDI following immunoprecipitation with anti-CD19 antibody in lysates of indicated transduced cell lines. The double CD19/CD81-KO Nalm6 cell line was used as negative control. E) Western blot for total levels of CD81, P97 and Actin in lysates from the indicated transduced cell lines. The double CD19/CD81- KO Nalm6 cell line was used as negative control. F) Immunoblotting of CD81 following immunoprecipitation with anti-CD19 antibody in lysates from primary patient samples expressing CD19-WT (101 and 105) and CD19-105R immunotherapy resistant isoform.

Article Snippet: 413 Antibodies 414 The following antibodies were used for protein detection: α-CD19-PE (Beckman Coulter #IM1285U), α-415 CD19 (Cell Signaling #3574), α-CD19 3G7, (Origen #TA506234), α-CD19 HD37 (Thermo Fisher #MA1-416 34005), α-VSVg-FITC (Abcam #ab3863), α-VSVg (Abcam # ab50549), α-CD81 (LifeSpan #LS-417 C359239), α-CD81-FITC (Molecular Probes #A15753), α-calnexin (Cell Signaling #2679), α-calreticulin 418 on A ugust 15, 2018 by guest http://m cb.asm .org/ D ow nloaded from (Cell Signaling #12238), α-UGCGL1 (NovoPro Labs # 116554), α-PDI (Cell Signaling #3501), α-Mouse 419 Alexa 488 (Thermo Fisher #R37114), α-rabbit Alexa 594 (Thermo Fisher #A-21207).

Techniques: Immunoprecipitation, Mass Spectrometry, Binding Assay, Staining, Flow Cytometry, Western Blot, Negative Control, Expressing

Journal: eLife

Article Title: Chimeric antigen receptors that trigger phagocytosis

doi: 10.7554/eLife.36688

Figure Lengend Snippet:

Article Snippet: Antibody , anti human CD19 (mouse antibody) , OriGene , TA506240 Clone OTI2F6 , IgG2a mouse monoclonal antibody.

Techniques: Cell Culture, Derivative Assay, Recombinant, Sequencing, Plasmid Preparation, Transduction, Titration, Purification, Electron Microscopy, Software

Journal: Journal of Translational Medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Transcription and expression of CD19 gene in the relapsed Nalm-6 cells. A Detection of CD19 transcripts in relapsed CD19 − Nalm-6 and wildtype Nalm-6 cells by TA cloning and sequencing. Sequences were compared to the reference sequence gene (NM_001770.6) and visualized by Exon-Intron Graphic Maker from WormWeb.org. Six alternatively spliced isoforms of CD19 mRNA were obtained and the percentages of them were shown separately, including the full-length (FL), skipping exon 2 (Δex2), partial deletion (partΔex2 and partΔex3), retention of intron 2 (in2) and combination of partΔex2 and in2. Of these, partΔex2, in2 and partΔex2 + in2 shift the reading frame, predicting that the expressed CD19 protein will be divided into three shortened proteins (purple, red and green) and a truncated CD19 protein (blue). B Schematic demonstration of single-cell purification of wild-type Nalm-6 cells (red) and relapsed CD19 − Nalm-6 cells (blue) by the limiting dilution method. C Flow cytometric profiles of membrane CD19 and cytosolic CD19 in subclones of Nalm-6 (red) and relapsed CD19 − Nalm-6 cells (blue). D Fluorescence microscopy of CD19 expression on the surface and cytoplasm using two anti-CD19 antibodies (HIB19, extracellular, green; D4V4B, intracellular, red) and DAPI (blue). E Immunoblotting for CD19 in protein lysates from wild-type Nalm-6 cells and relapsed CD19 − Nalm-6 cells using antibodies recognizing the extracellular domain (clone OTI5F3 from Origene, N-terminal) or the cytosolic domain (clone D4V4B from Cell Signaling, intracellular domain). F Expression of CD19 gene transcripts in RNA sequencing data from wild-type Nalm-6 and relapsed Nalm-6 subclones. G, H Volcano (G) and heatmap (H) plots demonstrating changes in differential gene expression of subclones measured by RNA sequencing. Differentially expressed gene (DEG) analysis identified 3453 DEGs with |log2Fc| ≥ 1 and p value < 0.05, and the heatmap for top 75 DEGs is shown

Article Snippet: E Immunoblotting for CD19 in protein lysates from wild-type Nalm-6 cells and relapsed CD19 − Nalm-6 cells using antibodies recognizing the extracellular domain (clone OTI5F3 from Origene, N-terminal) or the cytosolic domain (clone D4V4B from Cell Signaling, intracellular domain).

Techniques: Expressing, TA Cloning, Sequencing, Purification, Membrane, Fluorescence, Microscopy, Western Blot, RNA Sequencing Assay

Journal: Cancer cell international

Article Title: Syndecan-1 as an immunogene in Triple-negative breast cancer: regulation tumor-infiltrating lymphocyte in the tumor microenviroment and EMT by TGFb1/Smad pathway.

doi: 10.1186/s12935-023-02917-7

Figure Lengend Snippet: Fig. 5 A SDC1 expression in tumor cells associated with TILs in the TNBC cohort. a, b Patients with SDC1-positive expression and a low percentage of CD3+ TILs. c, d Patients with SDC1-positive expression and a low percentage of CD4+ TILs. e, f Patients with SDC1-positive expression and a low percentage of CD8+ TILs. g, h Patients with SDC1-positive expression and a low percentage of CD19+ TILs. B Frequencies of TILs between patients with SDC1-negative expression and SDC1-positive expression in tumor cells in the TNBC cohort. a Frequencies of TILs, b CD3+ TILs, c CD4+ TILs, d CD8+ TILs, e CD19+ TILs associated with SDC1 expression in tumor cells. C SDC1 expression in CAFs associated with TILs in the TNBC cohort. a, b Patients with SDC1-positive expression and a low percentage of CD3+ TILs. c, d Patients with SDC1-positive expression and a high percentage of CD4+ TILs. e, f Patients with SDC1-positive expression and a low percentage of CD8+ TILs. g, h The patients with SDC1-positive expression and a high percentage of CD19+ TILs. D Frequencies of TILs between patients with SDC1-negative expression and SDC1-positive expression in CAF in the TNBC cohort. a Frequencies of TILs, b CD3+ TILs, c CD4+ TILs, d CD8+ TILs, e CD19+ TILs associated with SDC1 expression in CAFs

Article Snippet: All TMA were stained for SDC1 (prediluted, Jinqiaoyatu, Beijing, China), CD3 (PA0553, prediluted; Leica Microsystems, Shanghai, China), CD4 (PA0427, prediluted; Leica Microsystems), CD8 (PA0183, prediluted; Leica Microsystems), CD19 (ZA-0569, Prediluted; Zhongshan Golden Bridge Biotechnology, Beijing, China).

Techniques: Expressing

Journal: International journal of molecular sciences

Article Title: Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes.

doi: 10.3390/ijms241310891

Figure Lengend Snippet: Figure 3. Real-time binding sensorgrams of the immobilized biotinylated anti-CD19 antibodies (ligand concentration 10 µg/mL) and CD19-AD2 construct (analyte). (A) FMC63-btn; (B) HIB19-btn; (C) 3B10-btn; (D) 4G7-btn were captured on SA biosensors and dipped in wells containing the analyte CD19-AD2 at different concentrations between 10 and 600 nM. The spectral shift, corresponding to the thickness of the biolayer, differs for individual analyses. The binding signals (blue sensorgrams) were obtained by subtracting the signals from ligand-coated SA biosensors dipped in wells with buffer (non-specific binding control). Fitted curves are depicted as red lines and were obtained by global fitting using a 1:1 ligand model.

Article Snippet: Binding kinetics of CD19-AD2 to various commercially available biotinylated antiCD19 antibodies, including FMC63 (Ab00613-2.0, Absolute Antibody Ltd., Oxford, Oxfordshire, UK), HIB19 (302204, BioLegend, USA), 4G7 (Ab00219-1.1, Absolute Antibody Ltd.), as well as 3B10 (TA506236AM, Origene Tech.

Techniques: Binding Assay, Concentration Assay, Construct, Control