Notch and EGFR Signaling Analysis

Protein extract preparation^{62 (link)}, immunoprecipitation assay^{63 (link)}, immunoblotting assays^{64 (link)}, and sucrose gradient for rafts isolation^{45 (link)} were performed as described elsewhere. Primary antibodies were as follows: anti-Notch3 (Catalog number 2889), anti-Notch1 (Catalog number 2421), anti-activated Notch1 (N1_Val1744), anti-EGFR D38B1 (Catalog number 4267S), anti-phospho-EGFR Y1173 (53A5-Catalog number 4407S), and anti-phospho-EGFR Y1068 (Catalog number 2234S), all from Cell Signaling (Danvers, MA, USA); anti-α-tubulin (Catalog number sc-8035), anti-Lamin B M20 (Catalog number sc-6217), anti-p27 C19 (Catalog number sc-528), anti-cyclin D1 M20 (Catalog number sc-718), anti-cyclin D3 C16 (Catalog number sc-182), anti-Notch1 L18 (N1_EC) (Catalog number sc-23299), anti-transferrin H65 (Catalog number sc-21011), and anti-PTPH1 (Catalog number sc-515181), all from Santa Cruz Biotechnology; anti-β-actin (Catalog number A5441) and anti-cholera toxin B subunit peroxidase conjugate (GM1, Catalog number C3741) from Sigma-Aldrich; and anti-Notch3 5E1 (N3_EC) antibody was kindly provided by Professor A Joutel⁶⁵.

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Diluvio G., Del Gaudio F., Giuli M.V., Franciosa G., Giuliani E., Palermo R., Besharat Z.M., Pignataro M.G., Vacca A., d’Amati G., Maroder M., Talora C., Capalbo C., Bellavia D, & Checquolo S. (2018). NOTCH3 inactivation increases triple negative breast cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest. Oncogenesis, 7(5), 42.

Publication 2018

anti-Notch3 anti-Notch1 anti-EGFR D38B1 anti-phospho-EGFR Y1173 anti-phospho-EGFR Y1068 anti-α-tubulin anti-Lamin B M20 anti-p27 C19 anti-cyclin D1 M20 anti-β-actin

Actin Antibodies Antibody Cholera Cyclin d1 Cyclin d3 Egfr Immunoprecipitation Lamin b Notch3 Peroxidase Protein Subunit Sucrose Toxin conjugate Transferrin α tubulin

Corresponding Organization :

Other organizations : Sapienza University of Rome, Karolinska Institutet, Novo Nordisk Foundation

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Variable analysis

independent variables

Protein extract preparation
Immunoprecipitation assay
Immunoblotting assays
Sucrose gradient for rafts isolation

dependent variables

Levels of Notch3, Notch1, activated Notch1, EGFR, phospho-EGFR Y1173, and phospho-EGFR Y1068
Levels of α-tubulin, Lamin B, p27, cyclin D1, cyclin D3, Notch1 (N1_EC), transferrin, and PTPH1
Levels of β-actin and cholera toxin B subunit (GM1)

control variables

Primary antibodies used for immunoblotting assays

positive controls

None specified

negative controls

None specified

Annotations

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