Western Blotting of Cell Signaling Proteins

Cell protein lysates were prepared, and Western blotting was performed according to our published protocols [34 (link), 105 (link), 107 (link)]. The following primary antibodies were used in this experiment: E2F1 (3742, Cell Signaling), E2F2 (sc-633, Santa Cruz Biotechnology), E2F3 (sc-878, Santa Cruz Biotechnology), cyclin B1 (sc-245, Santa Cruz Biotechnology), Mad2 (ab70383, Abcam), Bcl2 (2870, Cell Signaling), pBcl2 (Ser70) (2827, Cell Signaling), pSer/Thr/Phe (9631, Cell Signaling), cleaved caspase 3 (9661, Cell Signaling), cleaved PARP (5625, Cell Signaling), Hec1 (GTX70268, GeneTex, Irvine, CA), pBad (Ser136) (4366, Cell Signaling), Bad (9268, Cell Signaling), Mps1/TTK (3255, Cell Signaling), PP2Ac (2259, Cell Signaling), and Sgo1 (ab58023, Abcam). β-actin antibody (4970, Cell Signaling) was used as a loading control. For secondary antibodies, either goat anti-rabbit HRP (sc-2004) or goat anti-mouse HRP (sc-2005, Santa Cruz Biotechnology) were used. Signals were detected by using a Lumigen TMA-6 reagent (Lumigen Inc, Southfield, MI). Image J software (NIH, Bethesda, MD) was used to quantify protein levels.

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Lee M., Rivera-Rivera Y., Moreno C.S, & Saavedra H.I. (2017). The E2F activators control multiple mitotic regulators and maintain genomic integrity through Sgo1 and BubR1. Oncotarget, 8(44), 77649-77672.

Publication 2017

cyclin B1 Bcl2 pBcl2 (Ser70) cleaved caspase 3 cleaved PARP PP2Ac β-actin antibody goat anti-rabbit HRP goat anti-mouse HRP

Actin Antibodies Antibody Bcl2 Caspase 3 Cell Cyclin b1 E2f1 Goat Hec1 Mouse Mps1 Pbcl2 Protein Rabbit Sgo1

Corresponding Organization : Ponce Health Sciences University

Other organizations : Center for Cancer and Blood Disorders, Emory University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein levels of E2F1, E2F2, E2F3, cyclin B1, Mad2, Bcl2, phospho-Bcl2 (Ser70), phospho-Ser/Thr/Phe, cleaved caspase 3, cleaved PARP, Hec1, phospho-Bad (Ser136), Bad, Mps1/TTK, PP2Ac, and Sgo1

control variables

β-actin antibody used as a loading control

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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