Phosphor egfr

Manufactured by Cell Signaling Technology

Sourced in United States

Phosphor-EGFR is a laboratory product that detects and measures the phosphorylation of the epidermal growth factor receptor (EGFR) protein. It is a tool used in research applications to study cell signaling pathways involving EGFR activation.

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4 protocols using phosphor egfr

Detecting Signaling Pathway Activation

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Cell lysates were collected, and blotted as previously described [28 (link)]. c-Met (#8198), phospho-c-Met (Tyr1349; #3133), phospho-c-Met (Tyr1234/1235; #3077), Akt (#9272), phospho-Akt (Ser473; #9271), Erk1/Erk2 (#9107), phospho-Erk1/Erk2 (Thr202/204; #4376), EGFR (#2646), phospho-EGFR (Tyr1068; #3777), phosphor-EGFR (Tyr1173; #4407) ErbB3 (#4754), PARP (#9532) monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA). All antibodies were used at a 1:1000 dilution. -actin antibody (#A2228) was obtained from Sigma-Aldrich (St. Louis, MO) and was used at a 1:10,000 dilution.

Steinway S.N., Dang H., You H., Rountree C.B, & Ding W. (2015). The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma. PLoS ONE, 10(5), e0128159.

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Immunohistochemical Analysis of Ki67 and pEGFR

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The tumor tissues were fixed in 4% formalin for 24 h at room temperature, then dehydrated and embedded in paraffin. The 4-μm-thick sections were deparaffinized, rehydrated and covered with primary Ki67 (Proteintech Inc) and phosphor-EGFR (Cell Signaling Technologies) antibody at 4 °C overnight. Then, the samples were incubated with HRP-conjugated secondary antibody for one hour and DAB staining for detection. Finally, the positively stained cells were evaluated by digital image analysis with Image J software.

Fan F., Zhou M., Ye X., Mo Z., Ma Y., Luo L., Liang X., Liu H., Weng Y., Lin M., Liu X., Cai X, & Qian C. (2020). BEBT-109, a pan-mutant-selective EGFR inhibitor with potent antitumor activity in EGFR-mutant non-small cell lung cancer. Translational Oncology, 14(2), 100961.

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Comprehensive Phosphoproteomic Analysis of Cellular Signaling

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Cells were seeded in six-well plates and exposed to compounds at indicated concentrations for various times. The cells were collected and subjected to standard Western blot analysis as described previously 24 (link), with the antibodies against phosphor-AKT (Ser473), phosphor-AKT (Thr308), AKT, phosphor-ERK (Thr202/Tyr204), ERK, p110α, p110β, phosphor-PRAS40 (Thr246), PRAS40, Myc-tag, HRAS, phosphor-EGFR (Tyr1068), EGFR, IGF-1R, FGFR1, HER3, c-MET, HER2 (Cell Signaling Technology, Danvers, MA, USA), RAS (Merck Millipore, Billerica, MA, USA), V5-tag (Invitrogen, Carisbad, CA, USA). The β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Xu Y.C., Wang X., Chen Y., Chen S.M., Yang X.Y., Sun Y.M., Geng M.Y., Ding J, & Meng L.H. (2017). Integration of Receptor Tyrosine Kinases Determines Sensitivity to PI3Kα-selective Inhibitors in Breast Cancer. Theranostics, 7(4), 974-986.

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Western Blot Analysis of EGFR Signaling

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Cells were lysed in sodium dodecyl sulfate (SDS) lysis buffer (100 mM Tris-HCl, pH 7.5, 1% SDS, 10% glycerol) and boiled at 100 °C for 5 min, or lysed in TNE lysis buffer (1% NP-40, 10 mM Tris-HCl (pH 7.8), 0.5 M NaCl, 1 mM EDTA, Phostop, and Complete mini). Protein concentrations were measured using BCA Protein Assay Reagent (Thermo Fisher Scientific) after centrifugation at 20,000 g for 10 min. Equal amounts of protein from cell lysates were loaded on SDS-polyacrylamide gels for electrophoresis separation. Proteins were transferred to polyvinylidene difluoride membranes and immunoblotted with antibodies against phosphor-EGFR (Cell Signaling Technology, #4407 and #3777, 1:1,000), total EGFR (Santa Cruz Biotechnology, sc-03, 1:5,000, or Cell Signaling Technology, #4267), phospho-Akt (Ser473; Cell Signaling Technology, #4060, 1:1,000), total Akt (Cell Signaling Technology, #4691, 1:5,000), phospho-ERK (Cell Signaling Technology, #9101, 1:5,000), total ERK1/2 (Cell Signaling Technology, #9102, 1:5,000), phospoh-S6 (Ser240/244, Cell Signaling Technology, #5364, 1:8,000), total S6 (Cell Signaling Technology, #2217, 1:5,000), or β-actin (Sigma-Aldrich, A5228, 1:1,000). ECL Prime Western Blotting Detection Reagent (GE Healthcare) and Amersham Imager 600 (GE Healthcare) were used for signal detection.

Yoshizawa T., Uchibori K., Araki M., Matsumoto S., Ma B., Kanada R., Seto Y., Oh-hara T., Koike S., Ariyasu R., Kitazono S., Ninomiya H., Takeuchi K., Yanagitani N., Takagi S., Kishi K., Fujita N., Okuno Y., Nishio M, & Katayama R. (2021). Microsecond-timescale MD simulation of EGFR minor mutation predicts the structural flexibility of EGFR kinase core that reflects EGFR inhibitor sensitivity. NPJ Precision Oncology, 5, 32.

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