Western Blot Analysis of Protein Expression

For Western blot analyses, the indicated amounts of cell extracts were resolved in 10% SDS–PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, BioScience, Dassel, Germany). Membranes were blocked with 5% (wt/vol) nonfat dry milk in phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and probed with monoclonal anti-FLAG antibody (Sigma), monoclonal anti-APE1 antibody (Vascotto et al., 2009a (link)), monoclonal anti-Ku70 (sc-12729; Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal anti–RNA polymerase II (Abcam, Cambridge, MA), monoclonal anti-SIRT1 (Abcam), monoclonal anti-p32 (Santa Cruz Biotechnology), and polyclonal anti-p53(acetyl K382) (Abcam). Blots were developed by using the enhanced chemiluminescence procedure (GE Healthcare, Piscataway, NJ) or Western Lightning Ultra (Perkin Elmer, Waltham, MA). Data normalization was performed by using a monoclonal anti-tubulin antibody (Sigma). Blots were quantified by using a Chemidoc XRS video densitometer (Bio-Rad, Hercules, CA).

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Antoniali G., Lirussi L., D'Ambrosio C., Dal Piaz F., Vascotto C., Casarano E., Marasco D., Scaloni A., Fogolari F, & Tell G. (2014). SIRT1 gene expression upon genotoxic damage is regulated by APE1 through nCaRE-promoter elements. Molecular Biology of the Cell, 25(4), 532-547.

Publication 2014

monoclonal anti-FLAG antibody enhanced chemiluminescence procedure Western Lightning Ultra monoclonal anti-tubulin antibody Chemidoc XRS video densitometer

Anti antibody Ape1 Cell extracts Chemiluminescence Ku70 Membranes Milk Monoclonal antibody Nitrocellulose Phosphate Rna polymerase ii Saline Sds page Sirt1 Tubulin Tween 20 Western blot analyses

Corresponding Organization :

Other organizations : University of Udine, Istituto per il Sistema Produzione Animale in Ambiente Mediterraneo, National Research Council, University of Salerno, National Academies of Sciences, Engineering, and Medicine, Institute of Biostructure and Bioimaging

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Variable analysis

independent variables

Amounts of cell extracts

dependent variables

Protein expression levels of FLAG, APE1, Ku70, RNA polymerase II, SIRT1, p32, and acetylated p53

control variables

SDS-PAGE conditions (10% gel)
Transfer to nitrocellulose membranes
Blocking with 5% nonfat dry milk in PBS-Tween 20
Primary antibodies used (anti-FLAG, anti-APE1, anti-Ku70, anti-RNA polymerase II, anti-SIRT1, anti-p32, anti-acetylated p53)
Chemiluminescence detection method (ECL or Western Lightning Ultra)
Normalization using anti-tubulin antibody
Quantification using Chemidoc XRS video densitometer

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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