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Pegfr y1173

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PEGFR Y1173 is a lab equipment product that is used to detect and measure the phosphorylation of the tyrosine residue at position 1173 of the epidermal growth factor receptor (EGFR) protein. It is designed to facilitate research into cell signaling pathways and receptor tyrosine kinase activity.

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Lab products found in correlation

P akt, by Cell Signaling Technology (3 mentions) P erk1 2, by Cell Signaling Technology (2 mentions) Erk1 k23, by Santa Cruz Biotechnology (2 mentions) Akt 1 2 3 h 136, by Santa Cruz Biotechnology (2 mentions) P erk, by Cell Signaling Technology (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Caspase 3, by Cell Signaling Technology (1 mentions) Protran ba85, by GE Healthcare (1 mentions) Ecl reagent, by PerkinElmer (1 mentions) P gsk3β, by Cell Signaling Technology (1 mentions) α tubulin, by Merck Group (1 mentions) Versican v1, by Abcam (1 mentions) Versican v2, by Thermo Fisher Scientific (1 mentions) Twist, by Santa Cruz Biotechnology (1 mentions) Pegfr y845, by Cell Signaling Technology (1 mentions) Snail, by Cell Signaling Technology (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) N cadherin, by BD (1 mentions) Gsk3β, by BD (1 mentions) E cadherin, by BD (1 mentions)

Close spelling variants of this product

Anti-pEGFR (Y1173, (2 citations)

8 protocols using pegfr y1173

1

Immunoblotting Analysis of Protein Signaling

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Protein extracts were prepared as previously described [21] (link). 35 μg total proteins were denatured, separated on 10% SDS–PAGE, and transferred to nitrocellulose (Protran BA85, GE Healthcare Life Sciences). Membranes were incubated with primary antibodies diluted in NET-gelatin against pEGFR Y1173 (Cell Signaling Technologies, #4407), EGFR (Transduction Laboratories, E12020), pERK1/2 (Cell Signaling Technologies, #9101), ERK1 K23 (Santa Cruz, sc-94), pAKT (Cell Signaling Technologies, #9271), AKT1/2/3 H-136 (Santa Cruz, sc-8312), PARP (Cell Signaling Technologies, #9542), Caspase 3 (Cell Signaling Technologies, #9662), Tubulin (Sigma, T9026). Secondary HRP-conjugated anti-rabbit (Bio-Rad) and anti-mouse (Sigma) antibodies were used and detection was carried out using an ECL reagent (PerkinElmer, Rodgau, Germany) on X-ray films.
Xiao Z., Gaertner S., Morresi-Hauf A., Genzel R., Duell T., Ullrich A, & Knyazev P.G. (2017). Metformin Triggers Autophagy to Attenuate Drug-Induced Apoptosis in NSCLC Cells, with Minor Effects on Tumors of Diabetic Patients. Neoplasia (New York, N.Y.), 19(5), 385-395.
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2

Western Blot Analysis of EMT Markers

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Cultured cells were harvested by trypsin-EDTA, washed once with cold PBS and lysed in radioimmunoprecipitation (RIPA) buffer (Thermo scientific). Total proteins extracted from cells were analyzed on a 12% SDS-PAGE gel, and Western blot analysis was performed using antiserum against Versican V1 (Abcam; 1:1,000 dilution), Versican V2 (Thermo; 1:1,000 dilution), AKT (Cell Signaling; 1:1,000 dilution), pAKT (Cell Signaling; 1:1,000 dilution), ERK (Cell Signaling;1:1,000 dilution), pERK (Cell Signaling; 1:1,000 dilution), Snail (Cell-Signaling; 1:1,000 dilution), Twist (Santa Cruz; 1:1,000 dilution), E-cadherin (BD, 1:10,000 dilution), N-cadherin (BD; 1:1,000 dilution), GSK3β (BD; 1:1000 dilution), pGSK3β (Cell Signaling; 1:1,000 dilution), EGFR (Santa Cruz; 1:200 dilution), pEGFR (Y845) (Cell Signaling; 1:1,000 dilution), pEGFR (Y1173) (Cell Signaling; 1:1000 dilution), GAPDH (Santa Cruz; 1:1,000 dilution), and α-tubulin (Sigma; 1:1,000 dilution).
Lee H.C., Su M.Y., Lo H.C., Wu C.C., Hu J.R., Lo D.M., Chao T.Y., Tsai H.J, & Dai M.S. (2015). Cancer metastasis and EGFR signaling is suppressed by amiodarone-induced versican V2. Oncotarget, 6(40), 42976-42987.
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3

Quantifying Brain Tissue Markers

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Mouse brain tissue was fixed in 4% paraformaldehyde in PBS overnight and embedded in paraffin. Tissue sections (5 μm) underwent microwave antigen retrieval in a citrate buffer before immunostaining with the following antibodies: Ki-67 (Leica #NCL-Ki67p, 1:5000), cleaved caspase-3 (CC3) (BD #559565, 1:500), p-EGFR Y1173 (Cell Signaling #4407, 1:250), phosphorylated ErbB4 (pErbB4) Y984 (Cell Signaling #3790, 1:50), or total ErbB4 (Santa Cruz #SC-283, 1:200). Sections were developed using biotinylated secondary antibodies, followed by detection with an Elite ABC kit and NovaRED peroxidase substrate, then counterstained with Gill’s hematoxylin (all from Vector Laboratories). Ki67- and CC3-positive cells were quantified using a Nuance spectral unmixing camera and InForm Tissue Finder software (Perkin Elmer). Quantification of phosphorylated and total ErbB4 was performed using particle analysis in ImageJ [32] (link).
Endersby R., Whitehouse J., Hii H., Greenall S.A., Johns T.G, & Gottardo N.G. (2018). A Pre-Clinical Assessment of the Pan-ERBB Inhibitor Dacomitinib in Pediatric and Adult Brain Tumors. Neoplasia (New York, N.Y.), 20(5), 432-442.
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4

SAHA and LBH589 effects on EGFR

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LBH589 was provided by Novartis (Basel, Switzerland). Suberoylanilide hydroxamic acid (SAHA; also called vorinostat or zolinza) was purchased from Selleck Chemicals (Houston, TX). EGFR antibody (sc-71034) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). p-EGFR (Y1065) and p-EGFR (Y1173) antibodies were purchased from Cell Signaling Technology, Inc (#2234; Beverly, MA) and AbboMax (#600–290; San Jose, CA), respectively. The sources for other agents and antibodies were the same as described previously21 (link).
Zang H., Qian G., Zong D., Fan S., Owonikoko T.K., Ramalingam S.S, & Sun S.Y. (2020). Overcoming acquired resistance of EGFR-mutant NSCLC cells to osimertinib by combining osimertinib with the HDAC inhibitor, panobinostat (LBH589). Cancer, 126(9), 2024-2033.
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5

Cell Line Authentication and Kinase Inhibitor Assay

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LC-2/ad cells were obtained from Sigma (cat no. 94072247), TPC1 cells obtained from R.E. Schweppe (17 (link)); H2228 cells obtained from J.D. Minna. HCC78-TAER were previously described (18 (link)). Cells were maintained in RPMI-1640 (Invitrogen) with 10% FBS at 37°C in a humidified 5% CO2 incubator. Fingerprint analysis of cell lines was performed bi-annually by the Molecular Biology Service Center at the Barbara Davis Center for Diabetes at the University of Colorado Anschutz Medical Campus in Aurora, CO to ensure authenticity. Alectinib was provided by Chugai Pharmaceuticals. Ponatinib, cabozantinib, trametinib, gefitinib, afatinib, and foretinib were obtained from Selleck Chemicals. Pervanadate was generated by incubating hydrogen peroxide with 100 mM sodium orthovanadate in distilled water. Antibodies used were as follows: pEGFR Y1068 (D7A5), pEGFR Y1173 (53A5), total RET (D3D8R), pERK1/2 XP T202/Y204 (D13.14.4E), total ERK1/2 (L34F12), pAKT S473 XP (D9E), total AKT (40D4), and pSHC1 Y239/Y240 (2434) from Cell Signaling; pTYR (4G10 Platinum), GAPDH (6C5) and GAPDH (ABS16) from Millipore; pRET Y1062, α-tubulin (TU-02) and NRAS (F155) from Santa Cruz Biotechnology.
Nelson-Taylor S.K., Le A.T., Yoo M., Schubert L., Mishall K.M., Doak A., Varella-Garcia M., Tan A.C, & Doebele R.C. (2017). Resistance to RET-inhibition in RET-rearranged NSCLC is mediated by reactivation of RAS/MAPK signaling. Molecular cancer therapeutics, 16(8), 1623-1633.
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6

Fibroblast and Melanoma Cell Culture

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Fibroblasts NR6WT were cultured in alpha-MEM media supplemented with 1x non-essential amino acids, 1x L-glutamine, 1x sodium pyruvate, 1x pen/strep antibiotics, and 7.5% fetal bovine serum. Melanoma cell line WM1158 and ACTN4 knockdown WM1158 were cultured in DMEM (1 gL-1 glucose):L15 3:1 medium with 10% fetal bovine serum and 1x pen/strip antibiotics. Rat tail collagen I was purchased from BD Biosciences (San Jose, CA). Transfection reagent xFect was purchased from Clontech Life Technologies (Grand Island, NY). Monoclonal phosphor-tyrosine antibody (p-Tyr-100) and pEGFR (Y1173), polyclonal antibodies phosphor-p38, pERK, EGFR and pAKT (Ser473) were purchased from Cell Signaling Technology (Beverly, MA). Polyclonal Actin antibody was purchased from Sigma Aldrich (St. Louis, MO). Axl (Cat # SC-1097) and Tyro3 (cat # SC-1094) antibodies were purchased from Santa Cruz (Dallas, TA). Inhibitors PD153035, SB203580, PD98059, Ly294002 and FAK inhibitor II were purchased from MilliporeSigma-Calbiochem (Damstat, Germany). All inhibitors were used at a work concentration of 10 μM and cells were treated with them for 30 min.
Shao H., Wang A., Lauffenburger D, & Wells A. (2017). Tyro3-mediated phosphorylation of ACTN4 at tyrosines is FAK-dependent and decreases susceptibility to cleavage by m-Calpain. The international journal of biochemistry & cell biology, 95, 73-84.
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7

Western Blot Analysis of Cell Signaling

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Protein samples were electrophoresed using 7.5 or 10% SDS–PAGE and performed as previously described [45 (link)]. Membranes were incubated with the primary antibodies against pEGFR Y1173 (Cell Signaling, #4407), EGFR (Transduction Laboratories, E12020), pHER2 Y1248 (Cell signaling, #2247), HER2 (Millipore, #06-562), pHER3 Y1289 (Cell signaling, #4791), HER3 (Millipore, #05-390), pERK1/2 (Cell Signaling, #9101), ERK1 K23 (Santa Cruz, sc-94), pAKT (Cell Signaling, #9271), AKT1/2/3 H-136 (Santa Cruz, sc-8312), PARP (Cell Signaling, #9542), Tubulin (Sigma, T9026), Secondary HRP-conjugated anti-rabbit (Bio-Rad) and anti-mouse (Sigma) antibodies were used and detection was done using an ECL reagent.
Xiao Z., Sperl B., Gärtner S., Nedelko T., Stacher-Priehse E., Ullrich A, & Knyazev P.G. (2019). Lung cancer stem cells and their aggressive progeny, controlled by EGFR/MIG6 inverse expression, dictate a novel NSCLC treatment approach. Oncotarget, 10(26), 2546-2560.
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8

Inhibitor-based Signaling Pathway Analysis

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Growth factors were purchased from Life Technologies, Carlsbad, CA (EGF), and PeproTech, Rocky HIll, NJ (HGF, NRG-1, and IGF). The compounds used were Met inhibitor SU11274 (Selleck Chemicals, Houston, TX), EGFR inhibitor erlotinib (LC Labs, Woburn, MA; PeproTech), PTP1B inhibitor (539741; Calbiochem, Billerica, MA), and SHIP2 inhibitor AS1949490 (Tocris, Minneapolis, MN). The following antibodies were used: EGFR (Cell Signaling Technology, Danvers, MA), pEGFR Y1173 (Cell Signaling Technology; Epitomics), vimentin (BD Transduction Laboratories, San Jose, CA), E-cadherin (Cell Signaling Technology), PTP1B (Millipore, Burlington, MA), SHIP2 (Cell Signaling Technology), and panMena (Lebrand et al., 2004 (link)).
Hughes S.K., Oudin M.J., Tadros J., Neil J., Del Rosario A., Joughin B.A., Ritsma L., Wyckoff J., Vasile E., Eddy R., Philippar U., Lussiez A., Condeelis J.S., van Rheenen J., White F., Lauffenburger D.A, & Gertler F.B. (2015). PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena. Molecular Biology of the Cell, 26(21), 3867-3878.
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