Erbb3

Manufactured by Cell Signaling Technology

Sourced in United States

The ErbB3 is a protein that functions as a receptor for the epidermal growth factor (EGF) family of ligands. It is a member of the ErbB family of receptor tyrosine kinases and plays a role in cell signaling pathways.

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Lab products found in correlation

29 protocols using erbb3

Melanoma Cell Line Signaling Assay

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Antibodies including EGFR, ErBB2, ErBB3, ErBB4, pEGFR(Y1173), pEGFR(Y1068), pAkt(S473), Pan-Akt, pERK, ERK, pP38, pHSP27, GAPDH, and pTyr were purchased from Cell Signaling Technology (Danvers MA). Monoclonal antibody against E-cadherin was purchased from BD Biosciences (Franklin Lakes, NJ). Monoclonal GFP antibody was purchased from Santa Cruz Biotechnology (Dallas, TX). Recombinant human epidermal growth factor (rhEGF) was purchased from Millipore-Sigma (St Louis, MO). Transfection reagent Lipofectamine 2000 was purchased from Life Technologies (Grand Island, NY). Chemical inhibitors including PD153035, LY294002, and SB203580 were purchased from R&D Systems (Minneapolis, MN). MatriGel 24-well transwells were purchased from Corning (Corning, NY). Dox was purchased from Presenius Kabi (San Diego, CA). Melanoma cell lines WM983A, WM983B, IgR3, and WM852 were cultured in a medium containing three parts of DMEM (1 g L⁻¹ glucose) and one part of L15 in addition to 10% fetal bovine serum and 1× pen/strip antibiotics.

Shao H, & Wells A. (2023). Deciphering the molecular mechanism of enhanced tumor activity of the EGFR variant T790M/L858R using melanoma cell lines. Frontiers in Oncology, 13, 1163504.

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Protein Expression Analysis by Western Blot

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Whole-cell lysates were prepared using EBC lysis buffer (50-mM Tris–HCl [pH 8.0], 120-mM NaCl, 1% Triton X-100, 1-mM EDTA, 1-mM EGTA, 0.3-mM phenylmethylsulfonylfluoride, 0.2-mM sodium orthovanadate, 0.5% NP-40, and 5-U/mL aprotinin) and were then centrifuged. Proteins were separated using SDS-PAGE and transferred to PVDF membranes (Invitrogen) for Western blot analysis. Membranes were probed using antibodies against p-ALK (Tyr1604), ALK, p-Akt (Ser473), p-MET (Tyr1234/1235), p-ErbB2 (Tyr1221/1222), ErbB2, p-ErbB3 (Tyr1289), ErbB3, p-IGF1R (Tyr1135/1136), p-Erk (Thr202/Tyr204), caspase-3, PARP-1 (all from Cell Signaling Technology, Beverly, MA) Akt, Erk, p-EGFR (Tyr1173), EGFR, MET, and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA) as the first antibody; the membranes were then treated with a horseradish peroxidase-conjugated secondary antibody. All membranes were developed using an enhanced chemiluminescence system (Thermo Scientific, Rockford, IL). Densitometric analysis was performed using the ImageJ software provided by NIH (http://rsb.info.nih.gov/nih-image/).

Choi S.H., Kim D.H., Choi Y.J., Kim S.Y., Lee J.E., Sung K.J., Kim W.S., Choi C.M., Rho J.K, & Lee J.C. (2017). Multiple receptor tyrosine kinase activation related to ALK inhibitor resistance in lung cancer cells with ALK rearrangement. Oncotarget, 8(35), 58771-58780.

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Quantitative Immunoblotting of ErbB Phosphorylation

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Cells were lysed in situ in hot Laemmli sample buffer [132 (link)]. Cell lysates were further incubated for 5 minutes at 95°C. Primary antibodies were obtained from Abcam (Cambridge, MA, USA): phospho-ErbB3 Y1222 (#ab133445) and Y1289 (#ab76469) or Cell Signaling Technology (Danvers, MA, USA): ErbB1 (#4267), ErbB2 (#2165), ErbB3 (#12708), phospho-ErbB1 Y845 (#6963), Y992 (#2235), Y1068 (#3777), Y1086 (#2220), Y1148 (#4404), phospho-ErbB2 Y1196 (#6942), phospho-ErbB3 Y1197 (#4561), Y1328 (#8017) and actin (#3700). Secondary antibodies were derived from LI-COR Biosciences (Lincoln, NE, USA): IRDye 800CW goat anti-mouse IgG (#926-32210) and IRDye 800CW goat anti-rabbit IgG (#926-32211). Immunoblots were analyzed and quantified using the Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Jacobi N., Seeboeck R., Hofmann E., Schweiger H., Smolinska V., Mohr T., Boyer A., Sommergruber W., Lechner P., Pichler-Huebschmann C., Önder K., Hundsberger H., Wiesner C, & Eger A. (2017). Organotypic three-dimensional cancer cell cultures mirror drug responses in vivo: lessons learned from the inhibition of EGFR signaling. Oncotarget, 8(64), 107423-107440.

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Investigating EGFR Signaling Pathways

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Dulbecco’s Modified Eagle Medium (DMEM) with high glucose was from Invitrogen. The HCC827, H292, H4006, 293T cell lines were purchased from American Type Culture Collection. Enzastaurin and erlotinib were from LC Laboratories (Woburn, MA). Go6976 and U0126 were purchased from EMD Millipore (Billerica, MA). Anti-phospho-p70S6K (Thr389), S6K, mTOR, phospho- Erk½ (Thr202/Tyr204), Erk½, (E747-A750del Specific) EGFR, EGFR, P-Akt (Ser473), P-Akt (Thr308), Akt, PKCα and ErbB3 antibodies were from Cell Signaling Technology (Danvers, MA), and anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO). Anti-EEA1 antibody was from BD Biosciences (San Jose, CA). siRNAs for PKCα and Gab1 were from Qiagen (Valencia, CA) and those for PLCγ1 were from ambion (life technologies). Dual mTOR inhibitor (KU-0063794) and Akt inhibitor (MK-2206 2HCl) were purchased from Selleckchem. All other reagents were from Sigma-Aldrich (St. Louis, MO).

Salama M.F., Liu M., Clarke C.J., Espaillat M.P., Haley J., Jin T., Wang D., Obeid L.M, & Hannun Y.A. (2019). PKCα is required for Akt-mTORC1 activation in Non-Small Cell Lung Carcinoma (NSCLC) with EGFR mutation. Oncogene, 38(48), 7311-7328.

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Antibody Evaluation for Signaling Pathways

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The antibodies used were obtained from the following suppliers: EGFR, EGFR-pY1068, ErbB3, ErbB3-pY1289, AKT, AKT-pS473, focal adhesion kinase (FAK), and FAK-pY397 were from Cell Signaling Technology (Danvers, MA, USA), Extracellular signal-related kinase (ERK) 1, ERK-pY204, and Integrin β1 were from Santa Cruz Biotechnology (Dallas, TX, USA), and α-tubulin was from Sigma-Aldrich.

Nozaki M., Yasui H, & Ohnishi Y. (2019). Ligand-Independent EGFR Activation by Anchorage-Stimulated Src Promotes Cancer Cell Proliferation and Cetuximab Resistance via ErbB3 Phosphorylation. Cancers, 11(10), 1552.

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Molecular Regulation of Cancer Cell Signaling

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TSA, PXD101 and erlotinib were purchased from Selleckchem (Houston, TX). MG132 and recombinant human EGF were purchased from Sigma-Aldrich (St Louis, MO) and ProSpecBio (East Brunswick, NJ), respectively. The Arf1 dominant negative and constitutively active constructs pcDNA3-HA-Arf1 DN-T31 N (Arf1DN) and pcDNA3-HA-Arf1-ActQ71L (Arf1CA) were purchased from Addgene (Plasmid #10833 and #10832). Antibodies that recognize acetyl-Histone H3 (Lys9/Lys14), acetyl-Histone H4 (Lys8), p-AKT (Ser473), AKT, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-STAT3 (Tyr705), STAT3, p-Src (Tyr416), Src, p-EGFR (Tyr845), EGFR, p-ErbB2 (Tyr1221/1222), ErbB2, p-ErbB3 (Tyr1289) and ErbB3, were purchased from Cell Signaling Technology (Beverly, MA). β-actin and PY20 antibodies were purchased from Sigma-Aldrich (St Louis, MO). CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) Kit was obtained from Promega (Madison, MI).

He L., Gao L., Shay C., Lang L., Lv F, & Teng Y. (2019). Histone deacetylase inhibitors suppress aggressiveness of head and neck squamous cell carcinoma via histone acetylation-independent blockade of the EGFR-Arf1 axis. Journal of Experimental & Clinical Cancer Research : CR, 38, 84.

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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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Molecular Mechanisms of ErbB3 Regulation

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Valproic acid and LY294002 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in ddH₂O or dimethyl sulfoxide (DMSO) to make a stock solution at 500 mmol/L or 20 mmol/L, respectively. All the stock solutions were stored at − 20 °C. Recombinant human NRG-1 protein ab50227 was product from abcam (Cambridge, MA, USA).
MISSION® Non-target shRNA, which does not target human and mouse genes, control vector (pLKO.1-ConshRNA), and pLKO.1 containing human ErbB3 shRNA (pLKO.1-ErbB3shRNA) were purchased from Sigma. The packaging plasmids psPAX2 and pMD2.G for lentiviral expression vector were from Addgene Inc. (Cambridge, MA, USA).
Antibodies were obtained as follows: EGFR, ErbB2, ErbB3, PARP, Cleaved Caspase-3 (Asp175) (5A1E), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, STAT3, P-STAT3 (Tyr705), p21, Cyclin D1, RAS, Ki67 (Cell Signaling Technology, Inc., Beverly, MA, USA); β-actin (AC-75) (Sigma). All other reagents were purchased from Sigma unless otherwise specified.

Lin T., Ren Q., Zuo W., Jia R., Xie L., Lin R., Zhao H., Chen J., Lei Y., Wang P., Dong H., Huang L., Cai J., Peng Y., Yu Z., Tan J, & Wang S. (2019). Valproic acid exhibits anti-tumor activity selectively against EGFR/ErbB2/ErbB3-coexpressing pancreatic cancer via induction of ErbB family members-targeting microRNAs. Journal of Experimental & Clinical Cancer Research : CR, 38, 150.

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Protein Extraction and Western Blot Analysis of Organoids

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Organoids were harvested using Cell Recovery Solution (Corning) on ice.
Organoids were lysed using boiling SDS-lysis buffer (50mM Tris-HCL [pH 7.4], 2%
SDS). Protein lysates were separated using 4-12% Bis-Tris NuPage gels (Life
Technologies). Isolation of protein from hN organoids following treatment with
MEK and AKT antagonists required time points of 48 hours or less in order to
obtain sufficient protein quantities. Western blots were probed with the
following antibodies: phospho-ERK1/2 (4370, Cell Signaling), pan-ERK1/2 (4695,
Cell Signaling), phospho-Akt (4060, Cell Signaling), pan-Akt (4685, Cell
Signaling), phospho-ribosomal S6 (4858, Cell Signaling), S6 Ribosomal Protein
(2317, Cell Signaling), pan-EGFR (Abcam, ab2430), phospho-EGFR (3777, Cell
Signaling) ERBB2 (4290, Cell Signaling), phospho-ERBB2 (2247, Cell Signaling),
ERBB3 (12708, Cell Signaling), phospho-ERBB3 (4791, Cell Signaling) Heat Shock
Protein 90 (07-2174, Millipore or 4874, Cell Signaling), actin (sc-1616, Santa
Cruz Biotechnologies) and Kras (sc-30, Santa Cruz Biotechnologies). Loading
control for western blot is Hsp90 unless otherwise indicated.

Ponz-Sarvise M., Corbo V., Tiriac H., Engle D.D., Frese K.K., Oni T.E., Hwang C.I., Öhlund D., Chio I.I., Baker L.A., Filippini D., Wright K., Bapiro T.E., Huang P., Smith P., Yu K.H., Jodrell D.I., Park Y, & Tuveson D.A. (2019). Identification of Resistance Pathways Specific to Malignancy Using Organoid Models of Pancreatic Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(22), 6742-6755.

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Investigating EGFR Signaling Pathways

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