Whole Cell Protein Extraction and Western Blot

To generate whole cell extracts, cells were lysed with E1A lysis buffer (20mM NaH2PO4, 150mM NaCl, 0.5% IGEPAL, 5mM EDTA, 50mM NaF, 30mM Sodium pyrophosphate, 10% Glycerol). The following antibodies were used to detect protein expression by Western blot, MYC, actin, p53, H2B (Santa Cruz), POLRMT, Total OXPHOS Cocktail (Abcam), Caspase-3 (Cell Signaling), PARP (Biovision), γH2A.X (Millipore), and ORC2 (BD Bioscience). Western blotting of mitochondrial isolates for the mitochondrial proteins POLRMT, TFAM, HSP60 and porin has been described previously [7 (link)].

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Oran A.R., Adams C.M., Zhang X.Y., Gennaro V.J., Pfeiffer H.K., Mellert H.S., Seidel H.E., Mascioli K., Kaplan J., Gaballa M.R., Shen C., Rigoutsos I., King M.P., Cotney J.L., Arnold J.J., Sharma S.D., Martinez U.E., Vakoc C.R., Chodosh L.A., Thompson J.E., Bradner J.E., Cameron C.E., Shadel G.S., Eischen C.M, & McMahon S.B. (2016). Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer. Oncotarget, 7(45), 72395-72414.

Publication 2016

POLRMT Total OXPHOS Cocktail Caspase-3 γH2A.X

Actin Antibodies Buffer Caspase 3 Cell extracts Cells Edta Glycerol Mitochondrial Mitochondrial proteins Nacl Orc2 Porin Protein Sodium pyrophosphate Tfam Western blot

Corresponding Organization : Thomas Jefferson University

Other organizations : University of Pennsylvania, Cold Spring Harbor Laboratory, Stony Brook University, UConn Health, Pennsylvania State University, Roswell Park Comprehensive Cancer Center, Dana-Farber Cancer Institute, Harvard University, Yale University

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Variable analysis

independent variables

Lysis buffer (E1A lysis buffer: 20mM NaH2PO4, 150mM NaCl, 0.5% IGEPAL, 5mM EDTA, 50mM NaF, 30mM Sodium pyrophosphate, 10% Glycerol)

dependent variables

Protein expression levels of MYC, actin, p53, H2B, POLRMT, Total OXPHOS Cocktail, Caspase-3, PARP, γH2A.X, ORC2, TFAM, HSP60, and porin

control variables

Not explicitly mentioned

controls

Positive control: Western blotting of mitochondrial isolates for the mitochondrial proteins POLRMT, TFAM, HSP60 and porin as described previously [7].
Negative control: Not specified

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