Western Blot Analysis of Signaling Pathways

Western blotting was performed as described elsewhere [28 (link)]. Briefly, 5 milligrams of normal lung tissue from control mice (n=11) and 10% flaxseed-fed mice (n=9) were lysed, proteins were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes before probing with primary antibodies (1:1000; p-AKT, t-AKT, p-ERK, t-ERK, p-JNK, and t-JNK). Anti-rabbit HRP-conjugated secondary antibodies (1:1000) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific; Waltham, MA) were used to detect bands, and the images were captured using the MultiImage™ Light Cabinet (Alpha Innotech; San Leandro, CA). The densitometry results were obtained using ImageJ software. p-AKT, p-ERK, and p-JNK protein levels were normalized to their corresponding total proteins.

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Chikara S., Mamidi S., Sreedasyam A., Chittem K., Pietrofesa R., Zuppa A., Moorthy G., Dyer N., Christofidou-Solomidou M, & Reindl K.M. (2017). Flaxseed Consumption Inhibits Chemically-induced Lung Tumorigenesis and Modulates Expression of Phase II Enzymes and Inflammatory Cytokines in A/J Mice. Cancer prevention research (Philadelphia, Pa.), 11(1), 27-37.

Publication 2017

Anti-rabbit HRP-conjugated secondary antibodies SuperSignal West Pico Chemiluminescent Substrate MultiImage™ Light Cabinet

Anti antibodies Antibodies Densitometry Light Lung Membranes Mice Nitrocellulose Normal tissue P protein Polyacrylamide gels Proteins Rabbit

Corresponding Organization : North Dakota State University

Other organizations : HudsonAlpha Institute for Biotechnology, University of Pennsylvania

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Variable analysis

independent variables

Control mice (n=11)
10% flaxseed-fed mice (n=9)

dependent variables

P-AKT protein levels
T-AKT protein levels
P-ERK protein levels
T-ERK protein levels
P-JNK protein levels
T-JNK protein levels

control variables

Normal lung tissue from mice
Western blotting protocol as described elsewhere [28]
10% SDS-polyacrylamide gels
Nitrocellulose membranes
Primary antibodies (1:1000; p-AKT, t-AKT, p-ERK, t-ERK, p-JNK, and t-JNK)
Anti-rabbit HRP-conjugated secondary antibodies (1:1000)
SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific; Waltham, MA)
MultiImage™ Light Cabinet (Alpha Innotech; San Leandro, CA) for image capture
ImageJ software for densitometry analysis

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