Formalin-fixed and paraffin-embedded tissue blocks of the 50 PDX models were prepared. Candidate targets, including EGFR, HER3, MET, and PD-L1, were stained via immunohistochemistry (IHC) using anti-EGFR antibody (#4267, Cell Signaling Technology, Danvers, MA, USA), anti-HER3 antibody (#2708, Cell Signaling Technology), anti-MET antibody (#790-4430, Ventana Medical Systems, Tucson, AZ, USA), and anti-PD-L1 antibody (#M4420, Spring Bioscience Corp., Pleasanton, CA, USA) according to the manufacturers’ instructions. IHC results were evaluated according to a previously published method [11 (link)–13 (link)]. Fluorescent in situ hybridization (FISH) was performed for MET and EGFR genes using the MET/CEN7 Dual Color Probe Kit (Zytovision, Bremerhafen, Germany) and EGFR/CEN7 Dual Color Probe (Zytovision) according to the manufacturer’s instructions. Gene amplifications were defined as the ratio of MET/CEN7 ≥ 2.2 and EGFR/CEN7 ≥ 2.2. All of the IHC and FISH results were reviewed and scored by two independent pathologists blinded to each other.
Anti her3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-HER3 antibody is a laboratory research tool used to detect and quantify the presence of the HER3 (Human Epidermal Growth Factor Receptor 3) protein in various biological samples. HER3 is a member of the HER/ErbB family of receptor tyrosine kinases and plays a role in cell signaling pathways. The antibody specifically binds to the HER3 protein, allowing researchers to study its expression and distribution in their experiments.
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Lab products found in correlation
Anti egfr antibody, by Cell Signaling Technology (1 mentions)
Protein a agarose beads, by Merck Group (1 mentions)
Anti erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Halt protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti phospho erk1 2 antibody, by Cell Signaling Technology (1 mentions)
3 protocols using anti her3 antibody
1
Molecular Characterization of Patient-Derived Xenograft Models
2
Immunoprecipitation of p-EGFR and p-HER3
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A total of 1000 μg of protein was extracted from cells; then 1 μg of anti-HER3 antibody (Cell Signaling Technology) was added to the protein lysate and incubated for 1 hour at 4°C with gentle mixing. For each sample, 20 μl of Protein A Agarose beads (Sigma-Aldrich, St. Louis, MO) was added to 1.5-ml tubes and washed with lysis buffer four times before being added to the protein lysate containing the anti-HER3 antibody and incubated overnight at 4°C. The beads containing the antibody-target protein complexes were washed five times and then pelleted by centrifugation, SDS loading buffer was added, and samples were boiled for 5 minutes to separate beads and proteins. Beads were pelleted by centrifugation, and supernatants were collected and submitted to Western blot analysis for detection of p-EGFR as described above. EGFR pulled-down products were also collected to detect p-HER3 presence, and EGFR antibody for immunoprecipitation (IP) study was also purchased from Cell Signaling Technology.
3
Immunoblotting Analysis of Signaling Proteins
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Cells were treated with each substance and lysed with RIPA lysis buffer containing Halt Protease & Phosphatase Inhibitor Cocktail (78428, Thermo Fisher Scientific Inc.). The samples were loaded and separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The membranes were blocked and probed overnight with anti-phospho-HER3 antibody (#4791, Cell Signaling Technology), anti-HER3 antibody (#12708, Cell Signaling Technology), anti-phospho-AKT antibody (T40067, Abmart), anti-AKT antibody (T55561, Abmart), anti-phospho-ERK1/2 antibody (#4370, Cell Signaling Technology), anti-ERK1/2 antibody (#4695, Cell Signaling Technology), anti-β-tubulin antibody (R20005, Abmart) at 4°C. Then the membranes were washed and incubated with HRP-labeled secondary antibodies for 1 hour and visualized using the luminescent analyzer Tanon 4600 (Tanon, Inc).
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