Western Blotting of Cellular Fractions

Western blotting of cell body or neurite homogenates were performed as described previously.^{27 (link)} In addition to NAMPT (1 : 2000, Enzo Life Sciences, Exeter, UK), mouse monoclonal anti-histone H1 (1 : 500; Millipore, Nottingham, UK), and mouse monoclonal anti β-actin (1 : 5000; Abcam, Cambridge, UK) were used as loading controls for the cell body and the neurite fraction, respectively. For quantification, western blotting for NMNAT2 (2.0 μg/ml, Abcam) was performed as described in.^{16 (link)} Western blotting band intensities were determined and analysed with the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Western blotting of HEK293T or PC12 cell homogenates were performed as described,^{27 (link)} and blots were probed with an anti-his antibody (Sigma, H1029).

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Di Stefano M., Nascimento-Ferreira I., Orsomando G., Mori V., Gilley J., Brown R., Janeckova L., Vargas M.E., Worrell L.A., Loreto A., Tickle J., Patrick J., Webster J.R., Marangoni M., Carpi F.M., Pucciarelli S., Rossi F., Meng W., Sagasti A., Ribchester R.R., Magni G., Coleman M.P, & Conforti L. (2014). A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration. Cell Death and Differentiation, 22(5), 731-742.

Publication 2014

mouse monoclonal anti β-actin NMNAT2 H1029

Actin Antibody Cell body Histone h1 Mouse Nampt Neurite Pc12 cell

Corresponding Organization :

Other organizations : Queen's Medical Centre, University of Nottingham, Babraham Institute, Marche Polytechnic University, University of Edinburgh, University of California, Los Angeles, Doheny Eye Institute, Università di Camerino

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Variable analysis

independent variables

Cell body or neurite homogenates

dependent variables

NAMPT protein levels
NMNAT2 protein levels

control variables

Histone H1 (for cell body fraction)
β-actin (for neurite fraction)

controls

Positive control: Western blotting of HEK293T or PC12 cell homogenates and probing with an anti-his antibody

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