Immunoblotting Analysis of Cellular Signaling

Whole-cell lysates were prepared for SDS-PAGE as described previously (11 (link)). Briefly, 10–20 μg of protein were separated by SDS-PAGE, transferred to 0.45 μm polyvinylidene difluoride membranes (Bio-Rad), blocked with 5% nonfat dry milk in 1× TBS/0.1% Tween-20, and then probed with ezrin (Cell Signaling Technology, catalog no. 3145), radixin (Cell Signaling Technology, catalog no. C4G7), moesin (Cell Signaling Technology, clone Q480 catalog no. 3150), phospho-ezrin-radixin-moesin (Cell Signaling Technology, catalog no. 3726), phospho-Serine473 Akt (Cell Signaling Technology, catalog no. 9271), total Akt (Cell Signaling Technology, catalog no. 9272), phospho-Serine136 Bad (D25H8, Cell Signaling Technology, catalog no. 4366), total Bad (Cell Signaling Technology, catalog no. 9292), phospho-Serine536 p65 NFκB (Cell Signaling Technology, catalog no. 3031), total p65 NFκB (Cell Signaling Technology, catalog no. 8242), survivin (Cell Signaling Technology) or γ-tubulin (Sigma-Millipore, catalog no. T5326), with the appropriate secondary antibodies (Cell Signaling Technology).

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Hoskin V., Ghaffari A., Laight B.J., SenGupta S., Madarnas Y., Nicol C.J., Elliott B.E., Varma S, & Greer P.A. (2022). Targeting the Ezrin Adaptor Protein Sensitizes Metastatic Breast Cancer Cells to Chemotherapy and Reduces Neoadjuvant Therapy–induced Metastasis. Cancer Research Communications, 2(6), 456-470.

Publication 2022

polyvinylidene difluoride membranes ezrin radixin moesin phospho-ezrin-radixin-moesin phospho-Serine473 Akt total Akt total Bad total p65 NFκB survivin γ-tubulin

Antibodies Cell Clone Ezrin Membranes Milk Moesin Nfκb Polyvinylidene difluoride Protein Radixin Sds page Survivin Tubulin Tween 20

Corresponding Organization : Queen's University

Other organizations : Kingston Health Sciences Centre

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Variable analysis

independent variables

No independent variables are explicitly mentioned in the provided information.

dependent variables

Levels of ezrin
Levels of radixin
Levels of moesin
Levels of phospho-ezrin-radixin-moesin
Levels of phospho-Serine473 Akt
Levels of total Akt
Levels of phospho-Serine136 Bad
Levels of total Bad
Levels of phospho-Serine536 p65 NFκB
Levels of total p65 NFκB
Levels of survivin
Levels of γ-tubulin

control variables

SDS-PAGE was performed as described previously (11)
Proteins were separated by SDS-PAGE, transferred to 0.45 μm polyvinylidene difluoride membranes, blocked with 5% nonfat dry milk in 1× TBS/0.1% Tween-20

controls

No positive or negative controls are explicitly mentioned in the provided information.

Annotations

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