phosphorylated protein levels  (Cell Signaling Technology Inc)

Journal: Cancer Research Communications

Article Title: HER2-selective and reversible tyrosine kinase inhibitor tucatinib potentiates the activity of T-DM1 in preclinical models of HER2-positive breast cancer

doi: 10.1158/2767-9764.crc-23-0302

Figure Lengend Snippet: FIGURE 6 The combined suppression of HER2 signaling by tucatinib and T-DM1 is associated with reduced tumor growth. A, ELISAs quantifying phosphorylation of signaling components downstream of HER2 in BT-474 cells (as percentage phosphorylated protein vs. untreated cells with SEMs). B, IHC images of pHER2, pHER3, pAKT, and pMEK staining of the BT-474 xenograft tumor model. Inlay images represent HER2-stained tumor

Article Snippet: Phosphorylated protein levels were normalized to total protein levels (ErbB2, #7310;HER3, #7888C; AKT, #7170C; MEK, #7165C all from CST) and quantified relative to untreated cells.

Techniques: Phospho-proteomics, Staining

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