Figure 4. Sirt1 deacetylates 9G8. (A) HA-9G8 was co-transfected with pcDNA3.1/SIRT1 or pcDNA3.1/H363Y or siRNA of Sirt1 into HEK-293T cells. The cell extract was incubated with anti-HA precoupled to protein G beads. The bound proteins were subjected to western blots using anti-HA or anti-acetylated-lysine. The data are presented as mean ± S.D. (n=3) and analyzed with one-way ANOVA. **, p<0.01, *, p<0.05. (B) Immunoprecipitated HA-9G8 as described in A was incubated with or without Sirt1 in the presence of NAD+ for 2 h at 37°C. The level of 9G8 acetylation was analyzed by western blot developed with anti-HA and anti-acetylated-lysine and is presented as mean ± S.D. (n=3) and analyzed with student t-test. **, p<0.01.