Research Paper Volume 1, Issue 1 pp 38—48

Figure 3. Functional interaction between dSir2 and Dmp53. (A) Endogenous dSir2 physically interacts with Dmp53. A FLAG-tagged version of wild type Dmp53 was expressed in females during the first 10 days of adult life using the ELAV-Switch driver. Head extracts were then immunoprecipitated with anti-FLAG antibody. Western blot analysis with an antibody against dSir2 shows efficient co-immunoprecipitation of endogenous dSir2 with the over expressed wild type Dmp53-FLAG construct. (B) Recombinant dSir2 deacetylates human substrates. Recombinant purified dSir2 was incubated with the indicated substrates (5μM) in triplicate and released fluorescence was measured as Relative Light Units. No deacetylation activity was observed when no NAD was added or the Sir2 inhibitor nicotinamide was added. Shown is a representative of at least three independent experiments. (C) Recombinant dSir2 deacetylates Dmp53-derived peptides. Recombinant purified dSir2 was incubated in triplicate with the indicated Dmp53-derived peptides. Deacetylation activity is dose-dependent and reaches saturation at higher substrate concentrations. The SLKK substrate gets deacetylated with similar efficiency as the human p53 peptide and about twice as efficiently as the LSLK peptide, suggesting substrate specificity of dSir2. The experiments shown are background corrected for non-NAD containing reactions. Shown are representatives of at least three independent experiments.