Mouse tissues were extracted from 3 month old male mice sacrificed by CO2 asphixation and immediately frozen in liquid nitrogen. Tissues were then extracted using TriReagent (Sigma) according to manufacturers established procedures. RNA precipitate was extensively washed in 80% EtOH, re-suspended in DEPC treated water and stored at −80°C. Protein precipitate was extensively washed with 0.3 M guanadininum hydrochloride, 95% ethanol and re-suspended in 8M urea, 1% SDS and stored at −80°C. Protein concentration was determined using the DC Protein Assay (BioRad) according to manufacturer's procedures.
