Fig. 2: HINT1 binds to and is deacetylated by SIRT1.

a, b Flag-HINT1 was overexpressed in HEK293T cells, and then the cells were treated with a NAM for 24 h or b TSA for 6 h at different concentrations as indicated. c Flag-HINT1 was coexpressed with CBP, wild-type SIRT1 (WT), or SIRT1-catalytic mutant (H363Y) in HEK293T cells. a–c The acetylation levels of Flag-bead-IPed HINT1 were determined by western blot analysis. d SIRT1 was knocked down by a lentiviral system (shSIRT1) in DLD1 cells. Acetylation levels of HINT1 IPed with its antibody were determined by western blot assay. e For the in vitro deacetylation assay, 1 μg of the acetylated HINT1 protein was incubated with recombinant SIRT1 WT or SIRT1 HY in the presence or absence of NAD+ (1 mM) at 37 °C for 2 h, followed by western blot analysis. f, g For the reciprocal interaction between HINT1 and SIRT1, Flag-HINT1 was coexpressed with HA-SIRT1 in HEK293T cells. SIRT1 or HINT1 was immunoprecipitated with HA (f) or Flag (g) antibody, respectively, following western blot analysis using antibodies as indicated. h Endogenous HINT1 or SIRT1 proteins in SW480 or DLD1 cells were immunoprecipitated and immunoblotted with antibodies as indicated.