Phospho egfr tyr1068

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-EGFR (Tyr1068) is a primary antibody that specifically recognizes the epidermal growth factor receptor (EGFR) when phosphorylated at tyrosine 1068. This antibody can be used to detect and quantify the activation of EGFR signaling pathways.

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Anti-phospho-EGFR (Tyr1068) Rabbit monoclonal phospho-EGFR (Tyr1068) Phospho-EGFR (Tyr1068) antibody Rabbit anti-phospho-EGFR (Tyr1068) Rabbit anti-phospho-EGFR (Tyr1068) (D7A5) XP Anti-phospho-EGFR Tyr1068 antibody Phospho-EGFR

24 protocols using phospho egfr tyr1068

Western Blot Analysis of EGFR and Associated Signaling Proteins

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The following antibodies were used: mouse monoclonal antibodies against phospho(Tyr1068)-EGFR (Cell Signaling Technology Inc.), STAT3, and β-actin (Santa Cruz Biotechnology Inc., CA); rabbit monoclonal antibodies against CD44, phospho(S473)-Akt (Abcam, Cambridge, UK), EGFR, phospho(Thr705)-STAT3, and HER2 (Cell Signaling Technology Inc.); and rabbit polyclonal antibodies against Akt and phospho(Tyr1221/1222) -HER2 (Cell Signaling Technology Inc.).
Western blotting was performed as previously described [27 (link)]. In brief, the proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and detected by using specific antibodies. The blots were developed by using HRP-conjugated secondary antibodies and enhanced chemiluminescence detection system (Amersham Life Science, Piscataway, NJ). The images of the bands were obtained by using an Amersham Imager 600 system (GE healthcare).

Kim Y.G., Yoon Y.N., Choi H.S., Kim J.H., Seol H., Lee J.K., Seong M.K., Park I.C., Kim K.I., Kim H.A., Kim J.S, & Noh W.C. (2017). Breast cancer stem cells in HER2-negative breast cancer cells contribute to HER2-mediated radioresistance and molecular subtype conversion: clinical implications for serum HER2 in recurrent HER2-negative breast cancer. Oncotarget, 9(5), 5811-5822.

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Western Blot Analysis of Cell Signaling

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Anti-TM4SF4 antibody for western blot analysis was purchased from Sigma-Aldrich. Antibodies against phospho-Ser473 AKT, AKT, phospho-ERK, ERK, phospho-NF-kappaB (p65), NF-kappaB, phospho-PI3K, PI3K, PTEN, phospho-Tyr1068 EGFR, EGFR, phospho-IGF1R beta (Tyr1131)/insulin receptor beta (Tyr1146), IGF-1R beta (111A9), and anti-beta-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-MMP-2, anti-MMP-7, and anti-MMP-9 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blot analysis was performed as described previously [37 (link)]. Antibodies against N-cadherin and E-cadherin were purchased from BD Biosciences.

Choi S.I., Kim S.Y., Lee J., Cho E.W, & Kim I.G. (2014). TM4SF4 overexpression in radiation-resistant lung carcinoma cells activates IGF1R via elevation of IGF1. Oncotarget, 5(20), 9823-9837.

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Comprehensive Western Blot Analyses

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For western blot, samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with various antibodies; anti-GAPDH (#2118), N-cadherin (#13116), E-cadherin (#3195), Vimentin (#5741), Slug (#9585), Snail-1 (#3879), ZEB1 (#3396), ZO-1 (#8193), phospho-Met (Tyr1349) (#3121), Ron (#4269), p β-catenin (Thr41 or Ser45) (#9565), β-catenin (#8480), S6 (#2217), total Akt (#9272), phospho-c-Myc (Thr58/Ser62) (#9401), Ki-67 (#9027), phospho-AKT (Ser473) (#9271), phospho-EGFR (Tyr1068) (#11862), LRIG1 (#12752), c-Met (#8198) and antibodies were purchased from Cell Signaling Technology. LRIG1 (G-20) (#sc-50075) and EGFR (#sc-03) antibodies were purchased from Santa Cruz Biotechnology. c-Myc antibody (clone 9E11) (#MS-127-P0) was purchased from Neomarkers. Fibronectin (#GTX112794), CD44 (#GTX102111) and Twist (#GTX127310) were purchased from Genetex (CA, USA). Actin (#A5441) and Tubulin (#T5168) were purchased from Sigma (MO, USA). All antibodies used horseradish peroxidase-conjugated secondary antibodies (Biorad), followed by developing with SuperSignal West chemicals (Pierce). An AlphaInnotech imaging station with FluorChem software was used to capture images. All data are representative of more than three independent experiments.

Yokdang N., Hatakeyama J., Wald J.H., Simion C., Tellez J.D., Chang D.Z., Swamynathan M.M., Chen M., Murphy W.J., Carraway KL I.I.I, & Sweeney C. (2015). LRIG1 opposes epithelial to mesenchymal transition and inhibits invasion of basal-like breast cancer cells. Oncogene, 35(22), 2932-2947.

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Antibody Characterization for Signaling Pathways

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SP600125 was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Antibodies against phospho-ERK 1/2 (Thr-202/Tyr204), phospho-p38 MAPK (Thr-180/Tyr-182), phospho-JNK 1/2 (Thr183/Tyr185) and phospho-EGFR (Tyr1068) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Monoclonal antibody against β-actin was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Antibody against γ-tubulin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). MUC5AC antibody was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Antibody against HO-1 was purchased from Abcam (UK).

Oniki K., Nohara H., Nakashima R., Obata Y., Muto N., Sakamoto Y., Ueno-Shuto K., Imafuku T., Ishima Y., Watanabe H., Maruyama T., Otake K., Ogata Y., Suico M.A., Kai H., Shuto T, & Saruwatari J. (2020). The DsbA-L gene is associated with respiratory function of the elderly via its adiponectin multimeric or antioxidant properties. Scientific Reports, 10, 5973.

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Colon Cancer Cell Line Characterization

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Colon cancer SW480, DLD-1, HCT-116, HT-29, RKO and Caco-2 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). DiFi cells were purchased from Shanghai Yan Cheng biological technology Co., Ltd. (Shanghai, China). SW480 cells were grown in Leibovitz’s L-15 medium (Gibco, Gaithersburg, MD, USA), DiFi cells were grown in McCoy’s 5A medium (Gibco, Gaithersburg, MD, USA), and all the other cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Gaithersburg, MD, USA). Media were supplemented with 10% fetal bovine serum (FBS). Cetuximab was obtained from Merck KgaA (Darmstadt, Germany). PHA-665752 and PP2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dasatinib was purchased from Selleck chemicals (Houston, TX, USA). Antibodies to MET, phospho-MET (Tyr1234/1235), EGFR, phospho-EGFR (Tyr1068), SRC, phospho-SRC (Y416), poly (ADP-ribose) polymerase (PARP), AKT, phospho-AKT (Ser473), phospho-ERK1/ERK2 (Thr202/Tyr204), Caspase-3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-Actin, anti-ERK, secondary goat anti-rabbit and goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Song N., Liu S., Zhang J., Liu J., Xu L., Liu Y, & Qu X. (2014). Cetuximab-Induced MET Activation Acts as a Novel Resistance Mechanism in Colon Cancer Cells. International Journal of Molecular Sciences, 15(4), 5838-5851.

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Targeted Protein Pathway Analysis

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Erlotinib and osimertinib were purchased from Selleck Chemicals (Houston, TX, USA). Crizotinib was from Sigma-Aldrich (St. Louis, MO, USA). Chloroquine was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibodies used for immunoblot analyses were the products of Cell Signaling Technology (Danvers, MA, USA); anti-eIF3c (#2068), EGFR (#4267), phospho-EGFR (Tyr1068) (#3777), Akt (#9272), phospho-Akt (Ser473) (#9271), ERK1/2 (#9102), phospho-ERK1/2 (Thr202/Tyr204) (#9101), Met (#8198), phospho-Met (Tyr1234/1235) (#3077), LC3B (#3868), and β-actin (#4970).

Shintani T., Higashisaka K., Maeda M., Hamada M., Tsuji R., Kurihara K., Kashiwagi Y., Sato A., Obana M., Yamamoto A., Kawasaki K., Lin Y., Kijima T., Kinehara Y., Miwa Y., Maeda S., Morii E., Kumanogoh A., Tsutsumi Y., Nagatomo I, & Fujio Y. (2018). Eukaryotic translation initiation factor 3 subunit C is associated with acquired resistance to erlotinib in non-small cell lung cancer. Oncotarget, 9(101), 37520-37533.

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EGFR-Akt Signaling Modulation

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CM cells were plated in 6-well plates. Twenty-four hours after plating, cells were added with DMSO or 20 µg/ml cetuximab, 2.5 µM gefitinib, with or without 20 ng EGF, and were analyzed following 24h incubation. Whole cell lysate preparation and western blotting were performed as previously described (104 (link)). Ten µg cell lysate were separated on Criterion TGX 4-15% gradient gels (BioRad) and blotted onto nitrocellulose membranes. Immunoblotting was performed using the following antibodies, under the manufacturer’s instructions: anti-phospho-Akt (Ser473) (#9271), -total-AKT (#9271), -phospho-EGFR (Tyr1068) (#3777), and -total-EGFR (#2232) antibodies from Cell Signaling Technology; anti-β-tubulin (H-235) (sc-9104), used as a loading control, from Santa Cruz Biotechnology. Revelation was performed using the Clarity Western ECL Substrate (BioRad) or the SuperSignal Femto reagent (Pierce) through Chemidoc XRS+ instrument (Biorad). Image analysis was performed with the Image Lab v6.1 (Biorad) software. The Student’s t-test for two tailed distributions was used to compare data. Differences were considered statistically significant when p≤ 0.05.

Muraro E., Montico B., Lum B., Colizzi F., Giurato G., Salvati A., Guerrieri R., Rizzo A., Comaro E., Canzonieri V., Anichini A., Del Vecchio M., Mortarini R., Milione M., Weisz A., Pizzichetta M.A., Simpson F., Dolcetti R., Fratta E, & Sigalotti L. (2024). Antibody dependent cellular cytotoxicity-inducing anti-EGFR antibodies as effective therapeutic option for cutaneous melanoma resistant to BRAF inhibitors. Frontiers in Immunology, 15, 1336566.

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Evaluating Compound-Induced Cell Growth Inhibition

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Example 101

Proliferation Inhibition Assays:

Cell growth inhibition was assessed by MTS assay for Ba/F3, DFCI58-229, and DFCI127c cells, or by Cell Titer Glo Luminescent Cell viability assay (Promega®) for DFCI362JC cells. 3000 Ba/F3 cells were seeded for per well in 96-well plates, and were exposed to representative compounds of the application (3.3 nM to 10 μM) for 72 hours. For DFCI58-229, DFCI127c, and DFCI362JC cells. 5000 cells were seeded per well. All experimental points included 6 to 12 wells. Data were normalized to untreated cells and displayed graphically using GraphPad Prism (GraphPad Software, Inc.). The growth curves were fitted using a nonlinear regression model with sigmoidal dose response.

Western Blot Analysis:

Cells were plated at 5×10⁵cells per well in 6-well plates and treated with representative compounds of the application the following day. After 6 hours of treatment, cells were washed with PBS and lysed with NP40 buffer (Calbiochem®) supplemented with Complete™ Mini protease inhibitor and PhosSTOP™ phosphatase inhibitors (Roche®). Lysates were separated by SDS-PAGE gel, transferred to nitrocellulose membranes, and probed with the following antibodies: phospho-EGFR(Tyr1068) (3777), total EGFR (2232), p-Akt(Ser473) (4060), total Akt (9272), p-ERK(Thr202/Tyr204) (4370), total ERK (9102) (Cell Signaling), and HSP90 (SC-7947) (Santa Cruz Biotechnology®).

US11186574B2. Inhibitors of EGFR and/or HER2 and methods of use (2021-11-30). DANA-FARBER CANCER INSTITUTE, INC. [US], None [US]. Inventors: John M. Hatcher [US], Nathanael S. Gray [US], Jaebong Jang [US], Dries De Clercq [US], Pasi Janne [US], Jamie A. Saxon [US], Michael Eck [US], David A. Scott [US], Alyssa Verano [US].

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Analyzing EGFR Signaling in Mouse Lung Tumors

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Mouse lung tumors were flash frozen in liquid nitrogen, crushed and lysed in RIPA buffer containing protease inhibitor. Equal amounts of total protein were separated by gel electrophoresis on Mini-PROTEAN GTX 4-20% gels (Bio-Rad), transferred onto nitrocellulose (Bio-Rad) and probed with the following antibodies: phospho-EGFR (Tyr1068) (Cell Signaling #3777), EGFR (Cell Signaling #2232), SPC (Abcam #90716), and β-actin (Santa Cruz #47778).

Robles-Oteiza C., Ayeni D., Levy S., Homer R.J., Kaech S.M, & Politi K. (2021). Elevated murine HB-EGF confers sensitivity to diphtheria toxin in EGFR-mutant lung adenocarcinoma. Disease Models & Mechanisms, 14(11), dmm049072.

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Comprehensive Western Blot Analyses

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