Phospho egfr y1068

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-EGFR Y1068 is a primary antibody that specifically recognizes the epidermal growth factor receptor (EGFR) when phosphorylated at tyrosine 1068. This antibody is useful for detecting the activation state of EGFR in various experimental systems.

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Phospho-EGFR (85 citations) Y1068 (8 citations) Anti-phospho EGFR (Y1068), (7 citations) Anti-phospho-EGFR Y1068 antibody (3 citations) Rabbit anti‐phospho‐EGFR (Y1068) (3 citations) EGFR-Y1068 (2 citations)

20 protocols using phospho egfr y1068

Western Blot Analysis of Signaling Proteins

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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 EGFR^L858R (#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.

Pirazzoli V., Ayeni D., Meador C.B., Sanganahalli B.G., Hyder F., de Stanchina E., Goldberg S., Pao W, & Politi K. (2015). Afatinib plus cetuximab delays resistance compared to single agent erlotinib or afatinib in mouse models of TKI-naïve EGFR L858R-induced lung adenocarcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(2), 426-435.

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Comprehensive Antibody Evaluation Protocol

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Unless otherwise specified, all chemicals in the study, including DMSO (dimethyl sulfoxide) and emetine, were purchased from Sigma (St Louis, MO, USA). Erlotinib, gefitinib, doxorubicin, and cephaeline were purchased from Cayman Chemical (Ann Arbor, MI, USA). KRIBB11 and osimeritnib were purchased from Selleckchem (Houston, TX, USA).
Antibodies against HSF1 (#12972), HSP27 (#2402), HSP90α (#8165), PARP (#8165), phospho-ERK (#9101), ERK (#4695), phospho-MEK (#9121), MEK (#9122), BCL2 (#15071), MCL1 (#4572), caspase-3 (#9662), cleaved caspase-3 (#9664), PARP (#9542), EGFR (#4267), phospho-EGFR (Y1068) (#3777), MET (#8198), HER2 (#4190), and vimentin (#5741) were purchased from Cell Signaling Technology (Danvers, MA, USA), and actin antibody (sc-47778) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against phospho-HSF1 (Ser326) (#ab76076), BAG3 (ab47124) and E-cadherin (ab15148) were purchased from Abcam (Cambridge, MA, USA). N-cadherin antibody (640920) was purchased from BD Bioscience (San Jose, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit, mouse anti-rat, and goat anti-mouse IgG antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA).

Lee S., Jung J., Lee Y.J., Kim S.K., Kim J.A., Kim B.K., Park K.C., Kwon B.M, & Han D.C. (2021). Targeting HSF1 as a Therapeutic Strategy for Multiple Mechanisms of EGFR Inhibitor Resistance in EGFR Mutant Non-Small-Cell Lung Cancer. Cancers, 13(12), 2987.

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EGFR and Renilla Luciferase Knockdown

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Oligomers of 97 base pairs targeting EGFR and Renilla luciferase were subcloned into the modified retroviral mir-30 backbone miR-E (Fellmann et al., 2013 (link)). Retrovirus produced in Phoenix-E cells was used to transduce EO771 or LLC cells, which were selected for shRNA expression on the basis of puromycin resistance by culture in 8 µg/ml puromycin: EGFR, 5′-TGCTGTTGACAGTGAGCGACCACGAGAACTAGAAATTCTATAGTGAAGCCACAGATGTATAGAATTTCTAGTTCTCGTGGGTGCCTACTGCCTCGGA-3′; AREG, 5′-TGCTGTTGACAGTGAGCGATCAGAGGAGTATGATAATGAATAGTGAAGCCACAGATGTATTCATTATCATACTCCTCTGAGTGCCTACTGCCTCGGA-3′; and Renilla, 5′-TGCTGTTGACAGTGAGCGCAGGAATTATAATGCTTATCTATAGTGAAGCCACAGATGTATAGATAAGCATTATAATTCCTATGCCTACTGCCTCGGA-3′.
Knockdown of EGFR expression and signaling was verified by Western blot after 10-min stimulation of cells serum-starved overnight with recombinant mouse EGF or rmAreg (BioLegend) followed by lysis in 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, cOmplete protease inhibitor cocktail (Sigma-Aldrich), and PhosSTOP phosphatase inhibitor cocktail (Sigma-Aldrich). Lysates were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against β-actin (Sigma-Aldrich), phospho-Akt S473, phospho-EGFR Y1068, and phosphor-ERK1/2 T202/Y204 (Cell Signaling Technology).

Green J.A., Arpaia N., Schizas M., Dobrin A, & Rudensky A.Y. (2017). A nonimmune function of T cells in promoting lung tumor progression. The Journal of Experimental Medicine, 214(12), 3565-3575.

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Erlotinib Signaling Pathway Analysis

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Erlotinib (#S1023) was obtained from Selleck Chemicals (Houston, TX, USA). Puromycin (#A1113803) was purchased from Life Technologies (Carlsbad, CA, USA). Hoechst 33342 (#H21492) and propidium iodine (#P1304MP) were acquired from Invitrogen (Waltham, MA, USA). The Rictor (#2114), phospho-tyrosine (#5465), phospho-NDRG1 (T346; #5482), NDRG1 (#9485), phospho-Akt (S473; #4060), Akt (#9272), phospho-S6 (S240/4; #2215), S6 (#2217), phospho-4EBPl (T37/46; #2855), 4EBP1 (#9452), phospho-EGFR (Y1068; #3777), EGFR (#4267) and MET (#8198) antibodies were from Cell Signaling Technologies (Danvers, MA, USA). Antibodies against β-actin (#A1978) were purchased from Sigma (St. Louis, MO, USA).

Chiang C.T., Demetriou A.N., Ung N., Choudhury N., Ghaffarian K., Ruderman D.L, & Mumenthaler S.M. (2018). mTORC2 contributes to the metabolic reprogramming in EGFR tyrosine-kinase inhibitor resistant cells in non-small cell lung cancer. Cancer letters, 434, 152-159.

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Saracatinib Signaling Pathway Analysis

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Saracatinib was obtained from LC Laboratories. Antibodies for EGFR (no. 2232), phospho-EGFR^Y1068 (no. 3777), EGFR (no. 4267), HER2 (no. 2165), phospho-HER2^Y1221/1222 (no. 2243), AKT (no. 4685), phospho-AKT^S473 (no. 4060), Src (no. 2109), phospho-Src^Y416 (no. 6943), ERK1/2 (no. 4696) and phospho-ERK1/2^T202/Y204 (no. 4370) were obtained from Cell Signaling Technology; GADPH (sc-47724) from Santa Cruz Biotechnology; mouse anti-ee antibody (no. 901801) from Biolegend.

Lyu C., Bhimani A.K., Draus W.T., Weigel R, & Chen S. (2023). Active Gαi/o mutants accelerate breast tumor metastasis via the c-Src pathway. bioRxiv.

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Investigating EGFR and FGF Signaling Pathways

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The following antibodies were used: phospho EGFR Y1068 (Abcam AB5644),
phospho EGFR Y1068 (Cell Signaling 3777), EGFR (Cell Signaling 2646), EGFR (Cell
Signaling 4267), phospho ERK1/2 T202/Y204 (Cell Signaling 9101), phospho ERK1/2,
T202/Y204 (Cell Signaling 4370), ERK1/2 (Cell Signaling 9102), phospho AKT S473
(Cell Signaling 4060), AKT1/2/3 (Santa Cruz sc-8312), BIM (Cell Signaling 2933),
Actin (Cell Signaling 4970), Actin-HRP conjugated (Cell Signaling 12262), FGFR1
(Cell Signaling 9740), and FGFR3 (Cell Signaling 4574), phospho FRS2α
Y436 (Cell Signaling 3861), E-Cadherin (Cell Signaling 3195), N-Cadherin (Cell
Signaling 13116), Zeb1 (Cell Signaling 3396), Vimentin (Cell Signaling 5741).
Gefitinib, WZ4002, AZD9291, and BGJ398 (all from Selleck) were dissolved in DMSO
to a final concentration of 10 mmol/liter and stored at −20 °C.
The 18 drugs tested in the long-term assay are listed in Supplemental Table 3.

Raoof S., Mulford I.J., Frisco-Cabanos H., Nangia V., Timonina D., Labrot E., Hafeez N., Bilton S.J., Drier Y., Ji F., Greenberg M., Williams A., Kattermann K., Damon L., Sovath S., Rakiec D.P., Korn J.M., Ruddy D.A., Benes C.H., Hammerman P.S., Piotrowska Z., Sequist L.V., Niederst M.J., Barretina J., Engelman J.A, & Hata A.N. (2019). Targeting FGFR overcomes EMT-mediated resistance in EGFR mutant non-small cell lung cancer. Oncogene, 38(37), 6399-6413.

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EGFR Signaling Pathway Evaluation

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Phospho-EGFR (Y1068) (#3777), EGFR (#2646), phospho-AKT (S473) (#4058), AKT (#4685), phospho-ERK1/2 (T202/Y204) (#4376), ERK1/2 (#4695), and vinculin (#13901) primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). vinculin was used as loading control. HRP-conjugated anti-rabbit (#111-035-003) and mouse (#115-035-003) secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, US). Cetuximab (A2000) was purchased from Selleckchem (Houston, TX, USA). The recombinant human AREG (262-AR-100) was purchased from R&D Systems.

Kim S.A., Park H., Kim K.J., Kim J.W., Sung J.H., Nam M., Lee J.H., Jung E.H., Suh K.J., Lee J.Y., Kim S.H., Lee J.O., Kim J.W., Kim Y.J., Kim J.H., Bang S.M., Lee J.S, & Lee K.W. (2021). Amphiregulin can predict treatment resistance to palliative first-line cetuximab plus FOLFIRI chemotherapy in patients with RAS wild-type metastatic colorectal cancer. Scientific Reports, 11, 23803.

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IHC Evaluation of Protein Expression

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Protein expression was detected by IHC in serial sections of formalin-fixed paraffin-embedded tumour samples according to the manufacturer's instructions for each antibody. The samples were stained with antibodies against total EGFR, phospho-EGFR (Y1068), total ALK, and phospho-ALK (Y1604) (Cell Signalling Technology), followed by incubation in rabbit monoclonal anti-human ALK antibody (#3633 WP1-01; clone D5F3) at a dilution of 1:100. The staining intensity was scored from 0 to 3+; tumours with no expression (0) were described as negative for ALK protein expression, while tumours scored as 1+, 2+, or 3+ were deemed positive [45 (link), 46 (link)], and the staining intensity was scored according to the following criteria: 3+, intense, granular cytoplasmic staining; 2+, moderate, smooth cytoplasmic staining; 1+, faint cytoplasmic staining in 10% of tumor cells; and 0, no staining [45 (link)], and high phosphorylation of proteins means IHC ++ or +++; low phosphorylation of proteins means IHC + or +/− NA, not available. -/NA, IHC negative because of not available testing [20 (link), 38 ].

Lou N.N., Zhang X.C., Chen H.J., Zhou Q., Yan L.X., Xie Z., Su J., Chen Z.H., Tu H.Y., Yan H.H., Wang Z., Xu C.R., Jiang B.Y., Wang B.C., Bai X.Y., Zhong W.Z., Wu Y.L, & Yang J.J. (2016). Clinical outcomes of advanced non-small-cell lung cancer patients with EGFR mutation, ALK rearrangement and EGFR/ALK co-alterations. Oncotarget, 7(40), 65185-65195.

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Gene Expression Analysis Protocol

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For gene expression analysis, total RNA was isolated (High Pure RNA Isolation Kit, Roche Diagnostics, Mannheim, Germany) and reversely transcribed into cDNA (Transcription High Fidelity cDNA Synthesis Kit, Roche Diagnostics) following the manufacturer's instructions. Quantitative PCR analysis was performed on a LC480 instrument (Roche Diagnostics) using SYBR Green 1 Master chemistry (Roche Diagnostics) as described previously.^{35 (link)} Primer sequences were HPV16-E6 5′-TTGCTTTTCGGGATTTATGC-3′ and 5′-CAGGACACAGTGGCTTTTGA-3′, HPV16-E7 5′-CAGCTCAGAGGAGGAGGATG-3′ and 5′-GCCCATTAACAGGTCTTCCA-3′ and human ACTB 5′-TCAGCTGTGGGGTCCTGT-3′ and 5′-GAAGGGACAGGCAGTGAG-3′.
Protein expression and phosphoepitopes were detected by immunoblotting, immunohistochemistry or flow cytometry following established protocols. Primary antibodies were: p53, phospho-ERK1/2^T202/Y204, ERK 1/2, phospho-EGFR^Y1068 (all from Cell Signaling Technology, Danvers, MA, USA), β-actin (C4, ICN, Irvine, CA, USA), EGFR/HER1-, HER2-, HER3- and HER4-Phycoerythrin (R&D Systems, Minneapolis, MN, USA).

Pogorzelski M., Ting S., Gauler T.C., Breitenbuecher F., Vossebein I., Hoffarth S., Markowetz J., Lang S., Bergmann C., Brandau S., Jawad J.A., Schmid K.W., Schuler M, & Kasper S. (2014). Impact of human papilloma virus infection on the response of head and neck cancers to anti-epidermal growth factor receptor antibody therapy. Cell Death & Disease, 5(2), e1091-.

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Immunoblotting of Apoptotic and Signaling Proteins

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Cells were lysed and immunoblotting was performed as previously described15 (link),16 (link) using antibodies against cleaved caspase-3 (Asp¹⁷⁵), actin, phospho-EGFR-Y¹⁰⁶⁸, EGFR, phospho-protein kinase B (AKT)-S⁴⁷³, pan-AKT, phospho-signal transducer and activator of transcription (STAT)3-Y⁷⁰⁵, STAT3, phospho-extracellular signal-regulated kinase (ERK)1/2-T²⁰²/Y²⁰⁴, and ERK1/2 (Cell Signaling Technology, St Quentin en Yvelines, France), PARP (SantaCruz Biotechnology, Heidelberg, Germany), acetylated α-tubulin and α-tubulin (Abcam, Paris, France), and p21^WAF1 (Merck Millipore, Molsheim, France).

Jeannot V., Busser B., Vanwonterghem L., Michallet S., Ferroudj S., Cokol M., Coll J.L., Ozturk M, & Hurbin A. (2016). Synergistic activity of vorinostat combined with gefitinib but not with sorafenib in mutant KRAS human non-small cell lung cancers and hepatocarcinoma. OncoTargets and therapy, 9, 6843-6855.

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