CD-1 male mice were used for the investigation of telomerase in the mouse brain. All animal procedures were approved by the animal experimentation ethics committee of our institute.
Preparation of nuclear and cytoplasmic proteins extracts
Brains were quickly removed from the skull, washed, and placed in a Ringer solution at 4°C. These were homogenized using a manual homogenizer (pestle B). The homogenates were centrifuged at 500 g at 4°C for 7 min and the pellets were subjected to nuclear and cytoplasmic extractions.
The cytoplasmic extract was prepared as previously described. Briefly, cells were resuspended in buffer A (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 1 mM EDTA) containing a mixture of protease inhibitors [final concentrations: 2 μg/ml aprotinin, 2 μg/ml antipain, 2 μg/ml leupeptin, 1 μg/ml pepstatin A, 2 μg/ml PMSF (phenylmethylsulfonyl fluoride)], and kept on ice for 15 min. Lysis was performed by passing the solution 10–15 times through a syringe with a 21G needle. The cytosolic fraction was obtained by centrifugation at 9,300 g at 4ºC for 7 min and the supernatant was collected.
The pellet from the cytoplasmic preparation was resuspended in CHAPS buffer, containing 10 mM Tris-HCl, 5 mM 2-β-Mercaptoethanol pH 7, 1 mM MgCl2, 1 mM EDTA, 0.1 mM PMSF, 0.5% CHAPS (3[(3Cholamidopropyl)dimethylammonio]-propanesulfonic acid), and 10% glycerol, followed by incubation at 4ºC for 30 min. The nuclear extract was centrifuged for 30 min at 9,300 g at 4ºC, and the supernatant was collected.
DNA-bound protein extract
The pellet of the nuclear extract was washed with CHAPS twice, each time incubating for 20 min in ice and centrifuged for 20 min at 9,300 g in 4ºC. Then CHAPS with 1 M NaCl was added and the sample was incubated on ice for 30 min and centrifuged for 20 min at 9,300 g in 4ºC. The supernatant contained the DNA-bound proteins. Total protein concentration was determined using the BIO-Rad protein assay kit (Bio-Rad Laboratories).
Separation of proteins by gel electrophoresis and detection
Protein extracts (as indicated by various experiments) derived from nuclear and cytoplasmic fractions were analyzed by polyacrylamide gel electrophoresis and western blotting as previously described, using either anti-hTERT mice IgG hTERT monoclonal antibody (1:1000 1531-1; Epitomics, CA), anti-β-actin (1:7000 Irvine, CA), anti-top1 (1:500 goat IgG sc-26167), anti-lamin B (1:1000 mouse IgG sc-365214) and anti-β-tubulin (1:1000 mouse IgG sc-58886) (Santa Cruz Biotechnology, CA), or anti-VDAC (1:2000 rabit IgG ab34726). The immunocomplexes were detected by enhanced chemiluminescence (Santa Cruz Biotechnology).
Telomerase activity was assessed as described. Briefly, protein extract (at the indicated quantity) was incubated with the reaction mixture (see buffer section) that contains the reaction substrate: TS primer (5′-AATCCGTCGAGCAGAGTT-3') for 45 min at 30°C followed by PCR assay with [α-P32] dCTP using CX primer (5'-CCCTTACCCTTACCCTTACCCTTA-3') or ACX (5' GCG CGG CTT ACC CTT ACC CTT ACC CTA ACC 3'). Internal standard primers used as a control: IS primer (5'-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3') and ISR primer (5'-ATCGCTTCTCGGCCTTTT-3'). For detection of telomerase products in the brain extracts, 32 PCR cycles were used, and the internal standard primers were diluted to a concentration of 5×10-15 M. The PCR products were separated on 12.5% polyacrylamide gel, and the radioactive products were detected with phosphoimager (Bio-Rad Laboratories) or by autoradiography using x-ray films.
Results are presented as the mean ± standard error of the mean of at least three independent experiments. The statistical significance of the results was obtained using the Student’s t-test or one-way ANOVA, using OriginPro7 software. Statistical significance was set at a confidence level of 0.05.