Western Blot Analysis of Cell Signaling

Whole cell lysates were prepared from cells and subjected to western blot analyses as previously described (26 (link)). Western blots were incubated with primary antibodies from Cell Signaling Technology [anti-CDK1 (catalog #77055), anti-ERα (anti-AR, anti-PSA (catalog #5365), and anti-c-Myc (catalog #9402)], from Santa Cruz Biotechnology (Dallas, TX, USA) [anti-GSK3 (catalog #sc-7291), anti-AR (catalog #sc-7305), and anti-ERα (catalog #sc-543)], from Invitrogen [anti-DNMT1 (catalog #PA5-80557)], from Millipore [phosphoserine(catalog #AB1603)] and from Sigma-Aldrich [anti-GAPDH (catalog #MAB374)]. Anti-pDNMT1 (S714) is an in-house antibody. Blots were then incubated with anti-rabbit or anti-mouse secondary antibody (catalog #31460 and 31430, respectively, Thermo Scientific, Waltham, MA, USA) at room temperature. Signals were detected using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific, catalog #34094), visualized using the LI-COR Odyssey System (LI-COR Biosciences, Lincoln, NE, USA), and quantified using the ImageJ software.

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Sharma V., Joshi J., Yeh I.J., Doughman Y., Blankenberg D., Wald D, & Montano M.M. (2022). Re-Expression of ERα and AR in Receptor Negative Endocrine Cancers via GSK3 Inhibition. Frontiers in Oncology, 12, 824594.

Publication 2022

anti-CDK1 anti-c-Myc anti-AR anti-ERα anti-DNMT1 anti-GAPDH anti-rabbit or anti-mouse secondary antibody SuperSignal™ West Femto Maximum Sensitivity Substrate LI-COR Odyssey System

Anti antibody Antibodies Antibody Cdk1 Cells Dnmt1 Gapdh Gsk3 Mouse Phosphoserine Rabbit Sensitivity Western blots

Corresponding Organization : University School

Other organizations : Cleveland Clinic Lerner College of Medicine

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Variable analysis

independent variables

Primary antibodies used for Western blot analyses

dependent variables

Protein expression levels of CDK1, ERα, AR, PSA, c-Myc, GSK3, DNMT1, phosphoserine, and GAPDH

control variables

GAPDH as a loading control
Positive and negative controls not explicitly mentioned

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