Animals were killed by cervical dislocation and liver was removed after different time intervals, as indicated above. The handling and sacrifice of the mice were done as per the guidelines issued by BARC (Bhabha Atomic Research Center) animal ethics committee. Animals (6 weeks old) were subjected to total body -radiation at a rate of 50 cGy/min by using a 60 Co Theratron Junior Teletherapy unit (Atomic Energy of Canada Ltd., Ottawa, Canada). Area of exposure was kept constant.
2.4. Sample preparation for NAMPT and NMNAT quantification
Liver tissue was rinsed at least twice with phosphate buffered saline. To 100 mg of incised tissue, 1 ml of cell lysis buffer (50 mM Tris–HCl; pH 7.5, 5 mM EDTA; 150mM NaCl; 0.1% lauryl sulfate, sodium salt in deionized water (DW); 0.5% deoxycholic acid in DW; 1% Igepal CA-630 in DW; protease inhibitor cocktail (Sigma–Aldrich, St. Louis, USA) was added followed by incubation for 15min. It was followed by transfer of sample, along with cell lysis buffer to pre-chilled micro- homogenizer where the tissue was homogenized. The lysed cells were centrifuged (12,000 × g, 10 min) and protein-containing supernatant was removed to a chilled test and kept on ice till used for NAMPT and NMNAT quantification.
2.5. NAMPT and NMNAT assay
NAMPT was measured by sandwich immunometric assay (Nampt (mouse/rat) Intracellular ELISA kit, Biovision research products, CA, USA). Briefly, it involved incubation of NAMPT containing sample with capture antibody and peroxidase labeled detection antibody to form a stable immunocomplex. The peroxidase bound to the complex was developed by tetramethylbenzidine as substrate. The photometrically determined color (at nm) was proportional to NAMPT protein concentration. Nampt level were expressed as picogram NAMPT protein per gram liver. NMNAT activity was determined by spectrophotometric continuously coupled NMNAT and alcohol dehydrogenase reactions as described earlier []. NMNAT coupled assay system contained . ml mM Hepes (pH .) containing mMMgCl , . ml . mM ATP, . ml mM NMN, . ml . mg/ml yeast alcohol dehydrogenase (ADH) ( units), . ml of ethanol and g of enzyme protein in ml reaction mixture. The reaction was started by addition of the substrate NMN. The assay was based on marked increase in absorption at nm, which occurs when ADH using ethanol as substrate further reduces NAD formed. From the slope of linear progress curve enzyme activity was calculated. Nmnat activity was expressed as international unit (IU) per gram liver. One international unit of activity is defined as equivalent to enzyme required to transform mol of NMN to NADH per minute under the condition of assay.
NAD+ and ATP quantification
NAD+ was measured in tissue by enzyme cycling method described earlier and modified slightly in our laboratory. Tissue (mg) was washed with PBS at ◦ C, minced and extracted twice with l of . N perchloric acid for min at ◦ C. The acid supernatant collected at g for min was neutralized with ml of N KOH; .M KPO, pH .. After h on ice, KClO was precipitated by centrifugation at × g for min at ◦ C and discarded; the supernatant was stored at −◦C until NAD+ analysis. NAD+ assay system contained in a total volume of l, l of sample (or – pmol standard NAD+ ) and l of reaction mixture to obtain final concentration of . M bicine, pH .;. M ethanol; . mM EDTA, . mg/ml BSA; . mM -[,-dimethylthiazol–yl]-,-diphenyltetrazolium bromide (MTT); . mM phenazine ethosulfate. The reaction was started by the addition of U of alcohol dehydrogenase ( l of . mg/ml in . M bicine, pH .). The color developed was measured spectrophotometrically at nm. Calibration standards of -NAD+ in the range of – g/ml were used. ATP was estimated in mice liver using ATP colorimetric/fluorometric assay kit (Biovision, USA). Briefly, tissue ( mg) was lysed in l of ATP assay buffer and centrifuged at , × g for min to pellet insoluble materials. To l of resulting supernatant, l of ATP reaction mix was added in a -well plate. After min incubation, absorbance was measured at nm. The concentration of ATP was calculated using ATP standard curve and expressed in micromoles.
Nuclear extracts were prepared using the nuclear extraction kit (Sigma–Aldrich Chemical Co.) according to manufacturer’s protocol. PARP activity was detected in the nuclear extract using the universal colorimetric PARP assay kit (Trevigen Inc., Gaithersburg, MD, USA) according to the manufacturer’s instructions. In brief, serial dilutions of the PARP enzyme were distributed into wells to generate a standard curve. The clarified nuclear extract ( l/well) was added to triplicate wells to determine cellular PARP activity. The reactions were allowed to proceed for h at room temperature. The plate was washed times with × phosphate-buffered saline (PBS) and then incubated for min with l/well Streptavidin-horseradish peroxidase (Strep-HRP) diluted : in × Strep-Diluent (Trevigen Inc., Gaithers-burg, MD, USA). The plate was washed times with × PBS prior to the addition of the HRP substrate. For colorimetric readout, l of TACS-Sapphire (Trevigen Inc.) was added to each well and incubated in the dark, at room temperature, for min. Development of the colorimetric reaction was stopped by the addition of an equal volume of . M HCl. This generated a yellow color that was read at nm. The results were expressed as units of PARP activity calculated per milligram of protein and normalized to equal concentrations of protein. Protein content was determined by the bicinchoninic acid assay using bovine serum albumin as standard (Sigma–Aldrich Chemical Co). PARG activity was detected in nuclear extract using the universal colorimetric PARG assay kit (Trevigen Inc., USA) according to the manufacturer’s instructions. In this assay, PARP catalyzes the poly(ADP-ribosyl)ation of the activated histones attached to the wells. Subsequently, the product is degraded by the addition of PARG and poly(ADP-ribose) content is measured at nm.
