Journal: PLoS ONE

Article Title: Phosphorylation Regulates SIRT1 Function

doi: 10.1371/journal.pone.0004020

Figure Lengend Snippet: A. Affinity-purified SIRT1 is phosphorylated, and can be dephosphorylated by λppase. Top panel, western blot of total SIRT1; middle panel, western blot of SIRT1 phosphorylated at the Cdk consensus site; bottom panel, ProQ-diamond stained blot showing total phosphorylated SIRT1. Numbers above the lanes indicate the relative doses of λppase used to treat FLAG-SIRT1 for 1 hour. Overnight treatment (O/N) was done with a 1/10 dilution of λppase. B. Schematic of the phosphorylated residues in SIRT1 identified by mass spectrometry, as shown in . P, phosphorylated residue. C. 3D structure prediction of full-length human SIRT1 with the positions of phosphorylated residues identified by mass spectrometry (red symbols). Amino terminal domain, green; catalytic core domain, blue; carboxy terminal domain, yellow. The Rossmann fold supporting the catalytic groove in the core domain is shown in red. The two mutated residues described in the text (Thr530 and Ser540) are indicated with asterisks.

Article Snippet: The following antibodies were used: Peroxidase-conjugated AffiniPure Goat-anti-Mouse IgG and biotinylated anti-Mouse light chain (Jackson ImmunoResearch, West Grove, PA, #115-035-100, 1∶10,000), Peroxidase-conjugated AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch #110-035-144, 1∶10,000), anti-Cdc2 (Cdk1) mAb (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8395, 1∶1,000), anti-Cdk2 pAb (Abcam, Cambridge, MA, ab7954, 1∶1,000), anti-Cdk4 pAb (Abcam ab2945, 1∶1,000), anti-Cdk6 mAb (Abcam ab3126, 1∶1,000), anti-cyclinA pAb (Abcam ab7956, 1∶1,000), anti-cyclinB1 pAb (Abcam ab7957, 1∶1,000), anti-FLAG M2 mAb (Sigma-Aldrich F3165, 1∶2,000), anti-SIRT1 N-terminal polyclonal antibody (Millipore #07-131, 1∶1,000), anti-SIRT1 C-terminal polyclonal antibody AS-16 (Sigma-Aldrich #S5313, 1∶1,000), anti-SIRT1 monoclonal antibody 2G1/F7 (Millipore #05-707, 1∶1,000), anti-phospho serine Cdk substrate antibody (Cell Signaling Technology, Danvers, MA, #2324, 1∶2,000), anti-GAPDH antibody (Abcam ab8245, 1∶5,000), and anti-pericentrin antibody (Covance, Richmond, CA, PRB-432C, 1∶300).

Techniques: Affinity Purification, Western Blot, Staining, Mass Spectrometry

Journal: PLoS ONE

Article Title: Phosphorylation Regulates SIRT1 Function

doi: 10.1371/journal.pone.0004020

Figure Lengend Snippet: Conservation of phosphorylated residues, cyclin recognition motifs, and Cdk substrate motifs among SIRT1 orthologs of 12 different species.

Article Snippet: The following antibodies were used: Peroxidase-conjugated AffiniPure Goat-anti-Mouse IgG and biotinylated anti-Mouse light chain (Jackson ImmunoResearch, West Grove, PA, #115-035-100, 1∶10,000), Peroxidase-conjugated AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch #110-035-144, 1∶10,000), anti-Cdc2 (Cdk1) mAb (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8395, 1∶1,000), anti-Cdk2 pAb (Abcam, Cambridge, MA, ab7954, 1∶1,000), anti-Cdk4 pAb (Abcam ab2945, 1∶1,000), anti-Cdk6 mAb (Abcam ab3126, 1∶1,000), anti-cyclinA pAb (Abcam ab7956, 1∶1,000), anti-cyclinB1 pAb (Abcam ab7957, 1∶1,000), anti-FLAG M2 mAb (Sigma-Aldrich F3165, 1∶2,000), anti-SIRT1 N-terminal polyclonal antibody (Millipore #07-131, 1∶1,000), anti-SIRT1 C-terminal polyclonal antibody AS-16 (Sigma-Aldrich #S5313, 1∶1,000), anti-SIRT1 monoclonal antibody 2G1/F7 (Millipore #05-707, 1∶1,000), anti-phospho serine Cdk substrate antibody (Cell Signaling Technology, Danvers, MA, #2324, 1∶2,000), anti-GAPDH antibody (Abcam ab8245, 1∶5,000), and anti-pericentrin antibody (Covance, Richmond, CA, PRB-432C, 1∶300).

Techniques:

Journal: PLoS ONE

Article Title: Phosphorylation Regulates SIRT1 Function

doi: 10.1371/journal.pone.0004020

Figure Lengend Snippet: A/B. NAD + -dependent deacetylase activity of SIRT1 in the presence of CIP (A) or λppase (B) with or without phosphatase inhibitors. Western blots below each bar graph indicate the amount of SIRT1 in the reaction (top panel) and the degree of SIRT1 phosphorylation represented by Cdk-pSer signal (bottom panel). C. Deacetylase activity of affinity-purified FLAG-SIRT1 in the presence (left) or absence (right) of NAD + . Blue bars, no nicotinamide added; red bars, + nicotinamide. *, p<0.005; **, p<0.02; ***, p<0.002; #, p<0.01; ##, p<0.001 (Student's t -test). Error bars indicate +/−SEM.

Article Snippet: The following antibodies were used: Peroxidase-conjugated AffiniPure Goat-anti-Mouse IgG and biotinylated anti-Mouse light chain (Jackson ImmunoResearch, West Grove, PA, #115-035-100, 1∶10,000), Peroxidase-conjugated AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch #110-035-144, 1∶10,000), anti-Cdc2 (Cdk1) mAb (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8395, 1∶1,000), anti-Cdk2 pAb (Abcam, Cambridge, MA, ab7954, 1∶1,000), anti-Cdk4 pAb (Abcam ab2945, 1∶1,000), anti-Cdk6 mAb (Abcam ab3126, 1∶1,000), anti-cyclinA pAb (Abcam ab7956, 1∶1,000), anti-cyclinB1 pAb (Abcam ab7957, 1∶1,000), anti-FLAG M2 mAb (Sigma-Aldrich F3165, 1∶2,000), anti-SIRT1 N-terminal polyclonal antibody (Millipore #07-131, 1∶1,000), anti-SIRT1 C-terminal polyclonal antibody AS-16 (Sigma-Aldrich #S5313, 1∶1,000), anti-SIRT1 monoclonal antibody 2G1/F7 (Millipore #05-707, 1∶1,000), anti-phospho serine Cdk substrate antibody (Cell Signaling Technology, Danvers, MA, #2324, 1∶2,000), anti-GAPDH antibody (Abcam ab8245, 1∶5,000), and anti-pericentrin antibody (Covance, Richmond, CA, PRB-432C, 1∶300).

Techniques: Histone Deacetylase Assay, Activity Assay, Western Blot, Affinity Purification

Journal: PLoS ONE

Article Title: Phosphorylation Regulates SIRT1 Function

doi: 10.1371/journal.pone.0004020

Figure Lengend Snippet: A. Western blots of Cdk-phosphorylated (top panel) and total SIRT1 (bottom panel) following treatment with increasing doses of lambda phosphatase (λppase). The upper band corresponds to the full-length SIRT1. B. Levels of SIRT1 phosphorylation at Cdk-specific sites in extracts used as substrates for in vitro kinase assays shown in C. C. Autoradiographs (top panels) and Coomassie blue-stained gels (bottom panels) of SIRT1 and histone H1 phosphorylated by the kinases indicated on left. 0, 0.01, 0.1, 1, relative doses of λppase used; %P, relative percentage of Cdk phospho-serine signal compared to control.

Article Snippet: The following antibodies were used: Peroxidase-conjugated AffiniPure Goat-anti-Mouse IgG and biotinylated anti-Mouse light chain (Jackson ImmunoResearch, West Grove, PA, #115-035-100, 1∶10,000), Peroxidase-conjugated AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch #110-035-144, 1∶10,000), anti-Cdc2 (Cdk1) mAb (Santa Cruz Biotechnology, Santa Cruz, CA, sc-8395, 1∶1,000), anti-Cdk2 pAb (Abcam, Cambridge, MA, ab7954, 1∶1,000), anti-Cdk4 pAb (Abcam ab2945, 1∶1,000), anti-Cdk6 mAb (Abcam ab3126, 1∶1,000), anti-cyclinA pAb (Abcam ab7956, 1∶1,000), anti-cyclinB1 pAb (Abcam ab7957, 1∶1,000), anti-FLAG M2 mAb (Sigma-Aldrich F3165, 1∶2,000), anti-SIRT1 N-terminal polyclonal antibody (Millipore #07-131, 1∶1,000), anti-SIRT1 C-terminal polyclonal antibody AS-16 (Sigma-Aldrich #S5313, 1∶1,000), anti-SIRT1 monoclonal antibody 2G1/F7 (Millipore #05-707, 1∶1,000), anti-phospho serine Cdk substrate antibody (Cell Signaling Technology, Danvers, MA, #2324, 1∶2,000), anti-GAPDH antibody (Abcam ab8245, 1∶5,000), and anti-pericentrin antibody (Covance, Richmond, CA, PRB-432C, 1∶300).

Techniques: Western Blot, In Vitro, Staining