Journal: Journal of ovarian research

Article Title: Proteomic analysis of exosomes secreted during the epithelial-mesenchymal transition and potential biomarkers of mesenchymal high-grade serous ovarian carcinoma.

doi: 10.1186/s13048-023-01304-0

Figure Lengend Snippet: Fig. 2 Characterization of EGF-stimulation. EGFR-signaling evaluation by (A) antibody-array (CAOV3 cells) and, (B) Western Blotting (OvCa cells) kept FBS- free for 18 h, followed by 15/30 minutes EGF-stimulation, showing MEK 1/2 total, phosphorylated MEK (Ser-217/Ser-221), p-44/42 MAPK (Erk 1/2) total, and phosphorylated p-44/42 MAPK (Erk 1/2) (Thr-202/Tyr-204). (C) evaluation of ROS (CM-H2DCFDA) and, (D) mitochondrial membrane potential (JC-1 aggregate/JC-1) of OvCa cells after EGF EMT-induction (mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Article Snippet: PathScan EGFR signaling array kit The PathScan EGFR signaling array kit (Cat#12622, Cell Signaling) was utilized, which contains fixed antibodies specific to phosphorylated proteins in a chemiluminescent sandwich immunoassay format.

Techniques: Ab Array, Western Blot, Membrane

Journal: Frontiers in immunology

Article Title: Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.

doi: 10.3389/fimmu.2023.1168444

Figure Lengend Snippet: FIGURE 1 Biochemical characterization of ErbB-specific BiXAb™. (A) Schematic illustration of the BiXAb™platform. (B) Representative SDS-PAGE analysis of the BiXAb™1Cetu-2Pertu-Fc and 2Pertu-1Cetu-Fc in non-reducing and reducing conditions. Proteins were stained with Coomassie brilliant blue. IgG1 was used as control and molecular weight markers are indicated. Other BiXAb™are in Supplementary Figure 1. (C) Representative results of ELISA to assess binding of 1Cetu-2Pertu-Fc, 2Pertu-1Cetu-Fc, 1Cetu, and 2Pertu to immobilized EGFR (top panel) and HER2 (bottom panel). See Supplementary Figure 2 for all antibodies used in this study. (D) EC50 values extracted from ELISA binding curves.

Article Snippet: ELISA quantification of RTK expression RTK expression was evaluated with a sandwich ELISA using the PathScan® Cell Signaling Array kits to quantify total EGFR (#7250), HER2 (#7310C) and HER3 (#7888C).

Techniques: SDS Page, Staining, Control, Molecular Weight, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Frontiers in immunology

Article Title: Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.

doi: 10.3389/fimmu.2023.1168444

Figure Lengend Snippet: FIGURE 3 ELISA to assess the simultaneous binding of (A) 2Trastu-1Cetu-Fc to immobilized HER2 and EGFR-His (left) and immobilized EGFR and HER2-His (right); (B) 3Patri-2Trastu-Fc to immobilized HER3 and HER2-His (left) and immobilized HER2 and HER3-His (right); (C) 3Patri-1Cetu-Fc to immobilized HER3 and EGFR-His (left) and immobilized EGFR and HER3-His (right); and (D) 3Patri-1Matu-Fc to immobilized HER3 and EGFR-His (left) and immobilized EGFR and HER3-His (right). The EC50 values calculated from the ELISA binding curves are indicated.

Article Snippet: ELISA quantification of RTK expression RTK expression was evaluated with a sandwich ELISA using the PathScan® Cell Signaling Array kits to quantify total EGFR (#7250), HER2 (#7310C) and HER3 (#7888C).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Frontiers in immunology

Article Title: Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.

doi: 10.3389/fimmu.2023.1168444

Figure Lengend Snippet: FIGURE 4 Comparison of the four lead BiXAb™vs 2MAbs and single parental antibodies (1MAbs). (A) Effect on the phosphoproteome. Cells were pre- stimulated with BiXAb™, 2MAbs or 1MAbs for 20 min before adding a mixture of NRG1 and EGF for 10 min. Activation rate is relative to the maximal (100%) phosphorylation obtained in NRG1/EGFR-stimulated cells without antibodies. HuIgG1 IRR and HLA-DR-CD5-Fc were used as negative controls for 1MAbs and BiXAb™, respectively. The tyrosine kinase inhibitors iMEK and iPI3K were used for comparison. (B) Feedback loop strengths by modular response analysis in the indicated pancreatic cancer cell lines. (C) Effect on ErbB expression. The indicated cell lines were incubated with the indicated antibodies for 6 h. After cell lysis, EGFR, HER2 and HER3 levels were evaluated by sandwich ELISA (RD Systems) using receptor-specific antibodies. The percentage of expression was quantified relative to negative control (culture medium alone). The NRG1+EGF mixture was used as positive control. (D) Effect on ADCC assessed by monitoring CD107a degranulation and IFNg secretion. The indicated cell lines were co-cultured with CD16-transfected NK92 cells and incubated with BiXAb™, 2MAbs or 1MAbs for 4 h. The percentage of effector NK cells positive for CD107a and/or IFNg was measured by flow cytometry. Rituximab was used as irrelevant antibody. Data are the mean ± SEM from three replicates. The NRG1 + EGF mixture was used as negative control of ADCC. (E) Effect on cell viability. BxPC3 cells were incubated with BiXAb™, 2MAbs or 1MAbs for 5 days, and cell viability was measured using the MTS proliferation assay. (F) Effect on cell apoptosis. The indicated cells were incubated with BiXAb™ or 2MAbs for 48 h, and then labeled with fluorescein-conjugated Annexin V and 7-AminoActinomycin-D before apoptosis measurement (%) by flow cytometry. Medium and staurosporine (3 h incubation) were used as negative and positive controls of apoptosis, respectively. HLADR-CD5-Fc was used as irrelevant BiXAb™. Caspase 3/7 activation is reported in Supplementary Figure 5.

Article Snippet: ELISA quantification of RTK expression RTK expression was evaluated with a sandwich ELISA using the PathScan® Cell Signaling Array kits to quantify total EGFR (#7250), HER2 (#7310C) and HER3 (#7888C).

Techniques: Comparison, Activation Assay, Phospho-proteomics, Expressing, Incubation, Lysis, Sandwich ELISA, Negative Control, Positive Control, Cell Culture, Transfection, Cytometry, Proliferation Assay, Labeling

Journal: Frontiers in Immunology

Article Title: Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer

doi: 10.3389/fimmu.2023.1168444

Figure Lengend Snippet: Biochemical characterization of ErbB-specific BiXAb™. (A) Schematic illustration of the BiXAb™ platform. (B) Representative SDS-PAGE analysis of the BiXAb™ 1Cetu-2Pertu-Fc and 2Pertu-1Cetu-Fc in non-reducing and reducing conditions. Proteins were stained with Coomassie brilliant blue. IgG1 was used as control and molecular weight markers are indicated. Other BiXAb™ are in Supplementary Figure 1 . (C) Representative results of ELISA to assess binding of 1Cetu-2Pertu-Fc, 2Pertu-1Cetu-Fc, 1Cetu, and 2Pertu to immobilized EGFR (top panel) and HER2 (bottom panel). See Supplementary Figure 2 for all antibodies used in this study. (D) EC50 values extracted from ELISA binding curves.

Article Snippet: RTK expression was evaluated with a sandwich ELISA using the PathScan ® Cell Signaling Array kits to quantify total EGFR (#7250), HER2 (#7310C) and HER3 (#7888C).

Techniques: SDS Page, Staining, Molecular Weight, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer

doi: 10.3389/fimmu.2023.1168444

Figure Lengend Snippet: ELISA to assess the simultaneous binding of (A) 2Trastu-1Cetu-Fc to immobilized HER2 and EGFR-His (left) and immobilized EGFR and HER2-His (right); (B) 3Patri-2Trastu-Fc to immobilized HER3 and HER2-His (left) and immobilized HER2 and HER3-His (right); (C) 3Patri-1Cetu-Fc to immobilized HER3 and EGFR-His (left) and immobilized EGFR and HER3-His (right); and (D) 3Patri-1Matu-Fc to immobilized HER3 and EGFR-His (left) and immobilized EGFR and HER3-His (right). The EC50 values calculated from the ELISA binding curves are indicated.

Article Snippet: RTK expression was evaluated with a sandwich ELISA using the PathScan ® Cell Signaling Array kits to quantify total EGFR (#7250), HER2 (#7310C) and HER3 (#7888C).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer

doi: 10.3389/fimmu.2023.1168444

Figure Lengend Snippet: Comparison of the four lead BiXAb™ vs 2MAbs and single parental antibodies (1MAbs). (A) Effect on the phosphoproteome. Cells were pre-stimulated with BiXAb™, 2MAbs or 1MAbs for 20 min before adding a mixture of NRG1 and EGF for 10 min. Activation rate is relative to the maximal (100%) phosphorylation obtained in NRG1/EGFR-stimulated cells without antibodies. HuIgG1 IRR and HLA-DR-CD5-Fc were used as negative controls for 1MAbs and BiXAb™, respectively. The tyrosine kinase inhibitors iMEK and iPI3K were used for comparison. (B) Feedback loop strengths by modular response analysis in the indicated pancreatic cancer cell lines. (C) Effect on ErbB expression. The indicated cell lines were incubated with the indicated antibodies for 6 h. After cell lysis, EGFR, HER2 and HER3 levels were evaluated by sandwich ELISA (RD Systems) using receptor-specific antibodies. The percentage of expression was quantified relative to negative control (culture medium alone). The NRG1+EGF mixture was used as positive control. (D) Effect on ADCC assessed by monitoring CD107a degranulation and IFNγ secretion. The indicated cell lines were co-cultured with CD16-transfected NK92 cells and incubated with BiXAb™, 2MAbs or 1MAbs for 4 h. The percentage of effector NK cells positive for CD107a and/or IFNγ was measured by flow cytometry. Rituximab was used as irrelevant antibody. Data are the mean ± SEM from three replicates. The NRG1 + EGF mixture was used as negative control of ADCC. (E) Effect on cell viability. BxPC3 cells were incubated with BiXAb™, 2MAbs or 1MAbs for 5 days, and cell viability was measured using the MTS proliferation assay. (F) Effect on cell apoptosis. The indicated cells were incubated with BiXAb™ or 2MAbs for 48 h, and then labeled with fluorescein-conjugated Annexin V and 7-AminoActinomycin-D before apoptosis measurement (%) by flow cytometry. Medium and staurosporine (3 h incubation) were used as negative and positive controls of apoptosis, respectively. HLADR-CD5-Fc was used as irrelevant BiXAb™. Caspase 3/7 activation is reported in Supplementary Figure 5 .

Article Snippet: RTK expression was evaluated with a sandwich ELISA using the PathScan ® Cell Signaling Array kits to quantify total EGFR (#7250), HER2 (#7310C) and HER3 (#7888C).

Techniques: Activation Assay, Expressing, Incubation, Lysis, Sandwich ELISA, Negative Control, Positive Control, Cell Culture, Transfection, Flow Cytometry, Proliferation Assay, Labeling