Protein Isolation and Analysis Protocol

Total protein (~20 μg) was isolated from cells following 72 h treatment or vehicle control (0.02% DMSO or ethanol) for protein analysis as previously describe [40 (link),41 (link)]. Tris-glycine gels were used for all analysis except for BRCA1, ATM and the respective loading control where Tris-acetate gels were used to accommodate higher molecular weights of these proteins. The following antibodies were used: Actin (#4967), ATM (#2873), BRCA1 (#9010), gamma-H2AX (#80312), Rad51 (#8875), total-H2AX (#7631), p62 (#2947) and LC3BII (#2775) were from Cell Signaling (Danvers, MA, USA); CYP1B1 (ab185954) was from Abcam; β-tubulin (T7816) was from Sigma; DNA/RNA damage antibody (SMC-155) was from StressMarq (Victoria, BC, Canada) and Actin (#47778) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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Bakewell S., Conde I., Fallah Y., McCoy M., Jin L, & Shajahan-Haq A.N. (2020). Inhibition of DNA Repair Pathways and Induction of ROS Are Potential Mechanisms of Action of the Small Molecule Inhibitor BOLD-100 in Breast Cancer. Cancers, 12(9), 2647.

Publication 2020

Actin BRCA1 gamma-H2AX Rad51 total-H2AX LC3BII β-tubulin (T7816)

Acetate Actin Antibodies Antibody Brca1 Cells Cyp1b1 Dmso Dna damage Ethanol Gamma Gels Glycine Proteins Tris Tubulin

Corresponding Organization : Georgetown University

Other organizations : Intezyne (United States)

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Variable analysis

independent variables

Treatment (72 h treatment or vehicle control (0.02% DMSO or ethanol))

dependent variables

Total protein (~20 μg) isolated from cells
Actin
BRCA1
Gamma-H2AX
Rad51
Total-H2AX
LC3BII
CYP1B1
β-tubulin
DNA/RNA damage

control variables

Tris-glycine gels used for all analysis except for BRCA1, ATM and the respective loading control where Tris-acetate gels were used to accommodate higher molecular weights of these proteins

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