Protocol detail

Immunoblotting Analysis of Signaling Proteins

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Immunoblotting was performed essentially as described^{7 (link)}, except that the Westar ηC Ultra 2.0 chemiluminescence substrate (Cyanagen, Bologna, Italy) was employed. Rabbit monoclonal anti-ATP1B3 and rabbit polyclonal anti-transferrin receptor antibodies were purchased from Abcam (Cambridge, England). Mouse monoclonal anti β-catenin antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit monoclonal PI3 Kinase p100α, rabbit polyclonal AKT, rabbit polyclonal phospho-AKT (Ser⁴⁷³), rabbit monoclonal GSK3β, rabbit polyclonal phospho-GSK3β (Ser⁹) and phospho-Histone H2A.X (Ser¹³⁹) antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Monoclonal anti-β-actin antibody was purchased from Sigma (St. Louis, MO, USA).

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Veschi S., Ronci M., Lanuti P., De Lellis L., Florio R., Bologna G., Scotti L., Carletti E., Brugnoli F., Di Bella M.C., Bertagnolo V., Marchisio M, & Cama A. (2020). Integrative proteomic and functional analyses provide novel insights into the action of the repurposed drug candidate nitroxoline in AsPC-1 cells. Scientific Reports, 10, 2574.

Publication 2020

Westar ηC Ultra 2.0 chemiluminescence substrate Mouse monoclonal anti β-catenin antibody rabbit polyclonal AKT rabbit monoclonal GSK3β phospho-Histone H2A.X (Ser139) Monoclonal anti-β-actin antibody

Actin Anti antibodies Anti antibody Antibodies Atp1b3 Chemiluminescence Gsk3β Histone h2a x Monoclonal antibody Mouse Pi3 kinase Rabbit Transferrin receptor β catenin

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Variable analysis

independent variables

Westar ηC Ultra 2.0 chemiluminescence substrate (Cyanagen, Bologna, Italy)

dependent variables

ATP1B3 protein levels
Transferrin receptor protein levels
β-catenin protein levels
PI3 Kinase p100α protein levels
AKT protein levels
Phospho-AKT (Ser473) protein levels
GSK3β protein levels
Phospho-GSK3β (Ser9) protein levels
Phospho-Histone H2A.X (Ser139) protein levels
β-actin protein levels

control variables

Immunoblotting procedure as described in the referenced study (Yeh et al., 2018)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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