Crushed tumors were lysed in ice-cold RIPA lysis buffer [50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM MgCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, protease and phosphatase inhibitor cocktail (Thermo Scientific)]. Equal amounts of total protein were separated by SDS-PAGE and probed as indicated. Signals were detected using either SuperSignal West Pico or Femto chemiluminescent substrates (Pierce Biotechnology). Antibodies for immunoblotting against EGFRL858R (#3197), phospho-EGFR-Y1068 (#2234), phospho-Erbb2-Y1248 (#2247), AKT (#2938), phospho-AKT (#4060), ERK1/2 (#9102), phospho-ERK (#4376), S6 (#2217), phospho-S6 (#5364), GAPDH (#2118) and the secondary anti-rabbit HRP antibody (#7074) were from Cell Signaling Technology (CST). Additional antibodies include: Erbb2 (Millipore, 06-562), and SPC (Abcam, #ab90716). All the antibodies were used at the dilutions suggested by manufacturer.
Egfrl858r
Manufactured by Cell Signaling Technology
Sourced in United States
EGFRL858R is a recombinant protein that represents the EGFR L858R mutant. It is a tool used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Egfr e746 a750del, by Cell Signaling Technology (4 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Erbb2, by Merck Group (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Phospho egfr y1068, by Cell Signaling Technology (1 mentions)
Phospho s6, by Cell Signaling Technology (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Apoptag red in situ apoptosis detection kit, by Merck Group (1 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
Epha2, by Thermo Fisher Scientific (1 mentions)
Biotin goat anti rabbit, by BD (1 mentions)
Proliferating cell nuclear antigen (pcna), by BD (1 mentions)
Retrievagen a ph 6.0, by BD (1 mentions)
Bx53 dp74, by Olympus (1 mentions)
9 protocols using egfrl858r
1
Western Blot Analysis of Signaling Proteins
2
Quantitative Analysis of Tumor Biomarkers
3
Immunohistochemical Analysis of EGFR and HPV
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Immunohistochemical studies for EGFR and HPV were performed on 6-μm-thick tissue sections using the following antibodies: anti-human EGFR rabbit monoclonal antibody (clone: SP84; #414R-14; CELL MARQUE, Rocklin, CA, USA), anti-HPV mouse monoclonal antibody (clone: K1H8; ab75574; abcam, Cambridge, UK), horseradish peroxidase-conjugated anti-rabbit IgG (H+L) goat polyclonal antibody (HISTOFINE #424134, Nichirei Corporation, Tokyo, Japan), and horseradish peroxidase-conjugated anti-mouse IgG (H+L) goat polyclonal antibody (HISTOFINE #424144, Nichirei Corporation).
EGFR mutation-specific immunohistochemical staining was performed on 29 cases. As primary antibodies, we used EGFR E746-A750del (#2085, Cell Signaling Technologies, Danvers, MA, USA) and EGFR L858R (#3197, Cell Signaling Technologies), which were manually applied to the slides. Stained sections were viewed with an Olympus BX53+DP74.
As controls for staining, benign conjunctival lesions were also stained for EGFR, and colon cancer samples were stained as a positive control.
EGFR mutation-specific immunohistochemical staining was performed on 29 cases. As primary antibodies, we used EGFR E746-A750del (#2085, Cell Signaling Technologies, Danvers, MA, USA) and EGFR L858R (#3197, Cell Signaling Technologies), which were manually applied to the slides. Stained sections were viewed with an Olympus BX53+DP74.
As controls for staining, benign conjunctival lesions were also stained for EGFR, and colon cancer samples were stained as a positive control.
4
Lung Tissue Analysis of EGFR-TKI Treatment
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The animals were sacrificed immediately after imaging at 24 hours of EGFR-TKI treatment. The lungs were perfused with cold PBS supplemented with phosphatase and kinase inhibitors. The left lung was flash-frozen in liquid nitrogen for immunoblot, and the rest of the lung tissue was inflated with 4% paraformaldehyde in PBS. The lungs were fixed in 4% paraformaldehyde overnight at room temperature and placed in 70% ethanol before paraffin embedding. Serial sections of the lungs were obtained. The sections were stained with hematoxylin and eosin and probed with TTF (Dako), EGFRL858R (Cell signaling Technology), pERK (Cell signaling Technology) and Ki67 (Abcam). TUNEL staining was performed using the Apoptag Peroxidase In situ Apoptosis detection kit (Millipore).
5
Lung Adenocarcinoma Molecular Profiling
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We examined 93 cases undergoing surgery for adenocarcinoma of the lung at Osaka University Hospital from 2014 to 2018. Of 93 cases, 30 were subjected to RNA sequencing and 63 were subjected to immunohistochemistry. No prior therapy was given in any case. Histologic subtypes were classified according to WHO criteria.8 Resected specimens were fixed in 10% formalin and processed for paraffin embedding. Specimens were stored at room temperature in a dark room. Specimens for evaluation were sectioned at 4 μm thickness and stained with H&E. In 63 cases subjected to immunohistochemistry, we evaluated EGFR and KRAS mutations. We carried out immunohistochemistry using EGFR E746‐A750del (1:100, number 2085; Cell Signaling Technology) and EGFR L858R (1:100, number 3197; Cell Signaling Technology) as primary Abs, based on the criteria shown in our previous paper.9 We undertook mutational analysis for KRAS mutation. In more than 95% of cases of KRAS‐mutated lung cancer, KRAS missense mutations are found in codons 12 and 13 in exon 2.10 Therefore, we examined the sequences of KRAS exon 2. Genomic DNA from paraffin‐embedded tissue was extracted and purified. Exon 2 of KRAS was amplified using PCR primers.11 Sequence analysis was then carried out. The study was approved by the Ethical Review Board of the Graduate School of Medicine, Osaka University (No. 16293).
6
Immunohistochemical Analysis of Lung Tissue
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Lung tissue from normal, tumor bearing untreated and treated animals was collected after sacrifice, fixed overnight in 4% paraformaldehyde and rehydrated in 70% ethanol until submission for paraffin embedding and sectioning at the Yale Pathology Tissue Services. Sections were stained with hematoxylin and eosin, CD3 (Spring Biosciences; 1:150), EGFRL858R (Cell Signaling; 1:400), FoxP3 APC-conjugated (eBioscience; 1:50), Ki-67 (BioLegend; 1:50) and Cytokeratin 7 (Abcam; 1:300) antibodies. Positive cells in a 40X field of view were manually counted using a plugin for ImageJ called Cell Counter. At least three representative tissue locations were used to quantify and values were averaged for each mouse.
7
Immunohistochemical Profiling of Lung Tissue
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Lung tissue was harvested and fixed in 4% formaldehyde for 24 h, transferred to PBS, and embedded in paraffin (FFPE) using established routine protocols of the Pathology Department, University Hospital Cologne. Three micrometre lung sections were deparaffinised, and immunohistochemistry was performed on the LabVision Autostainer 480S (Thermo Fisher Scientific, Waltham, MA, USA). Staining was performed using haematoxylin and eosin (H&E), as well as primary antibodies against EGFRL858R (Cell Signaling Technologies, 3197, Leiden, The Netherlands), CD3 (Thermo Fisher, RM-9107-S, Waltham, MA, USA), CD4 (Abcam, EPR19514, Cambidge, UK), CD8 (Abcam polyclonal, ab203035, Cambidge, UK), and CD45R (BD Biosciences, 550286, Franklin Lakes, NJ, USA). Subsequently, primary antibodies were detected using secondary Histofine Simple Stain (SHSS) detection kits (Medac, Wedel, Germany). Slides were scanned on the Leica SCN400 Slidescanner (Leica Biosystems, Deer Park, IL, USA).
8
EGFR Immunohistochemistry Protocol for Tumor Cells
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Immunohistochemical staining was performed on 24 cases. We used EGFR E746-A750del (catalog number 2085, Cell Signaling Technologies (CST), Danvers, MA, USA) and EGFR L858R (catalog number 3197, Cell Signaling Technologies (CST)) as primary antibodies that were manually applied to the slides. The technique was carried out on a Benchmark® GX (Ventana Medical Systems, Tucson, AZ, USA) automated stainer. Immunoreactivity was revealed with UltraView Universal DAB detection kit (Ventana Medical Systems) and slides were counterstained with hematoxylin. Positive and negative controls were used. Each slide was examined and scored independently by two pathologists (Table 3 ) [30 (link)]. A specimen was considered positive in the presence of nuclear and/or cytoplasmic immunostaining for EGFR in tumor cells. Any discordance was resolved by consensus after a joint review using a multihead microscope.
9
Quantitative EGFR Expression Analysis
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Immunohistochemistry assays were performed as described previously. 34 Briefly, tissue sections were incubated with mutant EGFR rabbit monoclonal antibody (EGFR E746-A750del, #2085, 1:100; EGFR L858R, #3197, 1:100; Cell Signaling Technology (CST) Inc., Danvers, Massachusetts). Positive EGFR expression was determined by the intensity of membrane and/or cytoplasm staining of tumor cells and the proportion of EGFR-positive cells in the tissue sections. 35 The specimens were divided into four grades (0, 1þ, 2þ, 3þ) and were scored as follows: 0 (no staining), 1þ (light yellow staining without clear granular staining or yellow staining with clear granular staining area <10%), 2þ (yellow staining with clear granular staining area !10% or brown staining with clear granular staining area <10%), or 3þ (brown staining with clear granular staining area !10%).
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