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

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: Fibroblasts were infected with TB40/E GFP virus at an MOI of 1 for 0–72 hpi, with 0 hpi being uninfected. (A) To measure EGFR surface levels, infected cells were stained with brilliant violet 421 conjugated α-EGFR antibody and analyzed by flow cytometry. Normalized geometric mean fluorescent intensity is shown. (B) Total EGFR levels were measured over a time course by immunoblotting. Blots were stained with α-EGFR, α-IE1/2 antibody, and α-Tubulin. Both surface (A) and total (B) EGFR levels were normalized to 0 hpi for statistical analysis. IE proteins serve as a control for infection and tubulin serves as a control for loading. (C) Relative EGFR mRNA levels were measured over a time course using quantitative reverse transcriptase PCR and SYBR green. EGFR transcripts are normalized to H6PD, cellular housekeeping control, at each timepoint. (A-C) Statistical significance was calculated by One-Way ANOVA with Tukey’s correction and represented by asterisks (*** p-values < 0.001). Graphs represent the means from 3 independent replicates with error bars representing SEM. (D) Subcellular localization of EGFR was monitored over a time course of infection. Nuclei, EGFR and IE2 are visualized by staining with DAPI, α-EGFR, and α-IE2 and confocal deconvolution microscopy.

Article Snippet: Phosphorylation levels were measured using the PathScan EGFR Signaling Antibody Array Kit from Cell Signaling as per manufacturer recommended protocol using protein lysates at a concentration of 1 mg/ml.

Techniques: Infection, Virus, Staining, Flow Cytometry, Western Blot, Control, Reverse Transcription, SYBR Green Assay, Microscopy

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: (A) Fibroblasts were serum starved for 24h and cells were then infected for 0–72 hpi. At each timepoint, infected cells were pulsed with 10 nM of EGF for 0–30 min, and lysed. Lysates were separated out on SDS-PAGE gel, transferred on PVDF membrane, and stained for α-EGFR, α-pEGFR (Y1068), α-pAKT (S472), α-pMEK1/2 (S217/221), α-IE1/2 antibody, and α-Tubulin. (B) The 15 min post EGF timepoint for all phosphorylation markers were normalized to uninfected cells and graphed to calculate statistics. Statistical significance was calculated by One-Way ANOVA with Tukey’s correction and represented by asterisks (* p-value < 0.05 and ** p-value < 0.01).Graphs represent the mean of three replicates and error bars represent SEM. (E) Lysates from the 15 min post EGF-pulse at each time point post infection was separated by SDS-PAGE, transferred on PVDF membrane, and stained for α-AKT, α-pAKT(S472), α-MEK1/2, α-pMEK1/2(S217/221), α-IE1/2 antibody, and α-Tubulin to analyze total levels of AKT and MEK1/2. (F) CD34 + cells uninfected or infected with WT CMV (MOI of 2) were fixed and permabilized at 48 hpi. Cells were stained with PE conjugated α-CD34 + , Alexa Fluor 350 conjugated α-pEGFR(y1068), Dylight 649 conjugated α-pAKT(S473), Alexa Fluor 647 conjugated pMEK1/2(S217/221), and PerCP-eFluor 710 conjugated pERK1/2(T202/Y204) and the geometric mean of fluorescence determined by flow cytometry. Bars represent the average fold change in the geometric means from two replicates. Error bars represent the range of the two replicates.

Article Snippet: Phosphorylation levels were measured using the PathScan EGFR Signaling Antibody Array Kit from Cell Signaling as per manufacturer recommended protocol using protein lysates at a concentration of 1 mg/ml.

Techniques: Infection, SDS Page, Membrane, Staining, Phospho-proteomics, Fluorescence, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: (A) Fibroblasts were infected with TB40/E GFP (MOI = 1) and put into serum-free media at 24hpi. Cells were stimulated with 10nM EGF at 48 hpi and cells were harvested between 1 and 24 hours post stimulation. Lysates were separated by SDS-PAGE, transferred and blotted with α- IE1/2, α- UL135 , α- UL138 , and α Tubulin. Protein levels from 4 replicates were normalized to no EGF treated control and 1h post EGF treatment is graphed. Statistical significance was calculated by student t-test; asterisks *** p-value < 0.001. Error bars represent SEM.(B) Graphical representation of putative EGR1 binding sites located within UL135 ORF starting at nt-306 and nt-896, in reference to UL135 start codon. P-values were calculated using PhysBinder prediction software. (C) A model depicting how EGFR signaling promotes EGR1 expression by either directing its expression through MEK/ERK signaling or by blocking FOXO1 suppression of EGR1 transcription though PI3K/AKT signaling. (D) CD34 + HPCs were infected with WT virus (MOI of 2) and treated with MEK/ERK (Binimetinib and SCH772984) or PI3K/AKT (LY294002 and MK-2206) inhibitors at 4 hpi. RNA was isolated at 48 hpi EGR1 measured by RT-qPCR relative to H6PD. Graph represents the average from two replicates with error bars that represent the range.

Article Snippet: Phosphorylation levels were measured using the PathScan EGFR Signaling Antibody Array Kit from Cell Signaling as per manufacturer recommended protocol using protein lysates at a concentration of 1 mg/ml.

Techniques: Infection, SDS Page, Control, Binding Assay, Software, Expressing, Blocking Assay, Virus, Isolation, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: Our data demonstrates that EGFR signaling promotes EGR-1 expression through MEK/ERK signaling pathways. EGR-1 stimulates UL138 expression and UL138 feeds back to sustain EGFR signaling. UL135 targets EGFR for turnover and CMV miR-US22 targets EGR1 to negatively regulate this cycle for reactivation.

Article Snippet: Phosphorylation levels were measured using the PathScan EGFR Signaling Antibody Array Kit from Cell Signaling as per manufacturer recommended protocol using protein lysates at a concentration of 1 mg/ml.

Techniques: Expressing, Protein-Protein interactions

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: Primer sequences.

Article Snippet: Phosphorylation levels were measured using the PathScan EGFR Signaling Antibody Array Kit from Cell Signaling as per manufacturer recommended protocol using protein lysates at a concentration of 1 mg/ml.

Techniques: Sequencing

Journal: PLoS Pathogens

Article Title: Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency

doi: 10.1371/journal.ppat.1008037

Figure Lengend Snippet: Antibody description and sources.

Article Snippet: Phosphorylation levels were measured using the PathScan EGFR Signaling Antibody Array Kit from Cell Signaling as per manufacturer recommended protocol using protein lysates at a concentration of 1 mg/ml.

Techniques: Concentration Assay, Western Blot