Plasmid containing the human SIRT1 promoter was kindly provided by K. Irani, University of Pittsburgh (Pittsburgh, PA). This plasmid consists of a fragment of the human SIRT1 promoter (–1266 to +137 relative to transcription start site) cloned into the pGL4.1 firefly luciferase reporter vector (Promega, Madison, WI; Yamamori et al., 2010 (link)). The human SIRT1 promoter carrying the mutation at nCaRE-B sequences was generated with a Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA), using the primers SIRT1-B mut, forward, 5′-TCATCTAGGTTTTATTTATATATTTTTTTGCTAAGGAGCGTCGCTCTTGCTGCCCAGGCTGGTGTG-3′, and SIRT1-B mut, reverse, 5′-CACACCAGCCTGGGCAGCAAGAGCGACGCTCCTTAGCAAAAAAATATATAAATAAAACCTAGATGA-3′.
Expression and purification of recombinant proteins from E. coli were performed as previously described (Vascotto et al., 2009b (link); Fantini et al., 2010 (link)).
Expression and purification of recombinant proteins from E. coli were performed as previously described (Vascotto et al., 2009b (link); Fantini et al., 2010 (link)).