Caspase-3 activity was determined with the colorimetric caspase-3 detection kit (Sigma). dAdo uptake was analyzed by incubating cells with 1 μCi [3H]dAdo (specific activity: 38.1 Ci/mmol; Moravek Biochemicals). Radioactivity in washed cells was quantified by LSC and dAdo uptake reported as pmol/mg of total protein concentration in extracts. dAdo-derived nucleotides were quantified by treating cells with 0.5 μCi radiolabeled [14C]dAdo (specific activity: 48.8 mCi/mmol; American Radiolabeled Chemicals, Inc.) and 50 μM dCF. Cellular nucleotides were extracted using ice-cold 60% methanol (44 (link)) and separated by TLC (SIL G plates, Macherey-Nagel) using a water/isopropanol/ammonium bicarbonate mixture (25%:75%:0.2 M). Chromatograms were developed by autoradiography. Spots were excised and radioactivity was quantified by LSC to calculate nucleotide as picomoles per milligrams of protein (SI Appendix ).
Protocol detail
Quantification of Cellular Nucleotide Metabolism
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Ammonium bicarbonate
Autoradiography
Caspase 3
Cellular
Cold
Colorimetric
Isopropanol
Methanol
Nucleotide
Protein
Radioactivity
Spots
Corresponding Organization :
Other organizations : University of Chicago
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Variable analysis
independent variables
- DAdo (1 μCi [3H]dAdo, specific activity: 38.1 Ci/mmol)
- DAdo-derived nucleotides (0.5 μCi radiolabeled [14C]dAdo, specific activity: 48.8 mCi/mmol, and 50 μM dCF)
- Caspase-3 activity
- DAdo uptake (pmol/mg of total protein concentration in extracts)
- Cellular nucleotides (picomoles per milligrams of protein)
- Protein concentration in extracts
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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