Figure 11.

Cysteine alkylation decreases SIRT1 protein levels and enzymatic activity. A) BEAS-2B cells were treated with NEM (50 or 100 μM) for 30 min or 3 h, and SIRT1 was immunoprecipitated from whole-cell extracts for measurement of deacetylase activity. B) Extracts were also used for immunoblot for SIRT1 protein levels. β-Actin was used as a loading control. C) Nonalkylated/free cysteine residues on immunoprecipitated SIRT1 were labeled using maleimide-PEO₂-biotin and visualized using immunoblot with streptavidin conjugated to horseradish peroxidase. Blots are representative of 3 separate experiments (n=3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls; ⁺P < 0.05 vs. NEM (50 μM) at 3 h.