It was recently shown that Sir2 protein levels decline in aging haploid cells [36]. To determine if Sir2 levels are reduced in aging diploid cells, we purified aged mother cells from liquid MEP cultures and prepared total protein extracts. When the MEP is activated, the time a mother cell spends in culture corresponds to her replicative lifetime [16]. Hence we can age-match different strains by aging these MEP cultures for equivalent periods of time (the strains analyzed here divide at the same approximate rate; data not shown). To confirm age matching between strains, purified 26-hour populations were stained with calcofluor white to count bud scars (see Figure 6 legend). Western blotting with an anti-Sir2 antibody confirmed that Sir2 protein levels were dramatically reduced in aging diploid cells when normalized to total protein (Figure 6A). Sir2 levels in aging populations had fallen 10-fold by 26 hours, and were further reduced by 50 hours. This same decline was also observed with a polyclonal antiserum raised to a different region of the Sir2 protein (data not show). However, this behavior was not a
