2.3. SIRT1 activity
2.4. Cell metabolites
Acid-soluble metabolites were extracted in ice cold 0.3 M perchloric Safinamide and measured by HPLC after extract neutralization with buffered 3 M KOH. The cellular content of ATP and other nucleotides was determined after conversion into fluorescent etheno-derivatives [24]. NAD+ and NADH were directly determined in the neutralized extract by detection at 260 nm after separation [25].
2.5. RNA interference
The sequences of siRNAs directed against rat SIRT1 and the catalytic subunits of AMPK (Obtained from Sigma–Genosys) were as follows: for SIRT1, 5′-CACCUGAGUUGGAUGAUAU-3′; for AMPKα1, 5′-CUUAUUGGAUUUCCGAAGUTT-3′; for AMPKα2, 5′-GACAUUAUGGCGGAGGUGUTT-3′. Control siRNA-A was purchased from Santa Cruz Biotechnology. Cells at 50% of confluence were transfected with a final concentration of 100 nM siRNA duplex for 24 h by Transfection reagent (Santa Cruz) according to manufacturer’s instructions. 24 h after transfection, cells were then treated with the designated drugs.
View User's Journal
The tooth sections were examined
 |
