Quantitative Western Blot Analysis

Western blots were conducted as previously described [68 (link)]. Immunostaining was performed using goat monoclonal antibodies against phospho-Akt (S473) (cell signaling #9271) and actin (1/1000, cell signaling) and a secondary polyclonal mouse anti-goat antibody HRP conjugate (1/2000, cell signaling). Blots were developed using HRP and chemiluminescent peroxidase substrate (#CPS1120, Sigma). Data were collected using a Geliance CCD camera (Perkin Elmer, Waltham, MA, USA) and analyzed using ImageJ software (NIH) [21 (link)].

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Hoarau-Véchot J., Blot-Dupin M., Pauly L., Touboul C., Rafii S., Rafii A, & Pasquier J. (2022). Akt-Activated Endothelium Increases Cancer Cell Proliferation and Resistance to Treatment in Ovarian Cancer Cell Organoids. International Journal of Molecular Sciences, 23(22), 14173.

Publication 2022

CPS1120 Geliance CCD camera

Actin Anti antibody Goat Monoclonal antibodies Mouse Peroxidase Western blots

Corresponding Organization : Weill Cornell Medical College in Qatar

Other organizations : Université Paris-Est Créteil, Hôpital Intercommunal de Créteil, Centre de Recherche Saint-Antoine, Inserm, Sorbonne Université, Centre National de la Recherche Scientifique, Hôpital Tenon, Centre Expert en Endométriose, Cornell University

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Variable analysis

independent variables

Phospho-Akt (S473) immunostaining

dependent variables

Phospho-Akt (S473) protein levels
Actin protein levels

control variables

Western blot protocol as previously described [68]
Antibody concentrations (goat monoclonal antibodies against phospho-Akt (S473) (1/1000) and actin (1/1000), and secondary polyclonal mouse anti-goat antibody HRP conjugate (1/2000))
HRP and chemiluminescent peroxidase substrate (#CPS1120, Sigma) for blot development
Geliance CCD camera (Perkin Elmer, Waltham, MA, USA) for data collection
ImageJ software (NIH) [21] for data analysis

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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