A, Coimmunoprecipitation of SIRT1 with total JNK1 and phospho-JNK1 after treatment with anisomycin or H₂O₂. Anisomycin and H₂O₂ cause phosphorylation of JNK1 and interaction with SIRT1. B, Coimmunoprecipitation of SIRT1 with either total JNK1 or phospho-JNK1 from HEK293 cell lysates. SIRT1 immunoprecipitated specifically with phospho-JNK1 in response to anisomycin. C, Coimmunoprecipitation of SIRT1 with either total JNK1 plus phospho-JNK1 or phospho-JNK1 from C2C12 cell lysates. SIRT1 immunoprecipitated with phospho-JNK1 in response to anisomycin or H₂O₂.

A, Inhibition of SIRT1 activity by nicotinamide or Sirtinol does not affect phosphorylation of JNK1 in response to anisomycin. B, Inhibition of SIRT1 activity by nicotinamide or Sirtinol does not affect activity of JNK1 in response to anisomycin. JNK1 activity was measured by its ability to phosphorylate c-Jun. C, Knockdown of SIRT1 by siRNA does not affect anisomycin-mediated phosphorylation of JNK1. D, Knockdown of SIRT1 does not affect anisomycin-stimulated activity of JNK1. Clear bars are control. Black bars are SIRT1 knockdown.

A and B, These peptide MSMS fragmentation spectra were obtained from the ex vivo FL hSIRT1 IP samples after SDS PAGE and in-gel digestion. The spectrum in A shows phosphorylation of pS27 with neutral loss of 98 Da from the y8*, y9* and y10* ions (indicated with a *). Spectrum B shows phosphorylation of pS47 with neutral loss of 98 Da from the b3* and b4* ions, but no neutral loss from the y ions. C, The MSMS spectrum shown here was derived from in vitro experiments with hSIRT1 (a.a. 170–701) and shows phosphorylation of pT530 with neutral loss of 98 Da for y14* to y17*. These spectra were produced from Mascot database searches with high confidence scores using data generated from a nanoLC LTQ FT mass spectrometer.

Immunohistochemistry of SIRT1 and JNK1. Cells were treated with control (A), H₂O₂ (B), JNKi (C) or H₂O₂ plus JNKi (D). Cellular localization of JNK1 was determined by anti-rabbit JNK antibody and SIRT1 by anti-mouse SIRT1 antibody. Cells were stained with DAPI stain to visualize nuclei. Arrows show SIRT1 localization.

A, Schematic representation of full length human SIRT1 (FL hSIRT1) protein and a truncated form (aa 170–701 hSIRT1). Triangles show putative JNK1 phosphorylation sites. B, phosphorylation of SIRT1 by recombinant JNK1. FL hSIRT1 or a.a. 170–701 hSIRT1 was incubated with recombinant JNK1 or GSK3β in the presence of γ-[³²P]-ATP. C, Schematic representation of mouse SIRT1 GST fragments. Triangle represents the putative JNK phosphorylation site. D, In vitro phosphorylation of mouse SIRT1 GST when treated with JNK1 immunoprecipitates from anisomycin-treated C2C12 cells or recombinant JNK1. Truncated hSIRT1 (a.a. 170–701 hSIRT1) was used as positive control.

A, In vitro phosphorylation of full length (FL) hSIRT1 or SIRT1 with the putative JNK phosphorylation sites mutated (Mt-SIRT1). FL hSIRT1 or Mt-SIRT1 was incubated with recombinant JNK1. B, Mt-SIRT1 shows decreased activity in deacetylation of histone H3. Cells were transfected with the empty vector (CMV), WT hSIRT1 (WT) or Mt-SIRT1 (Mt) then treated with H₂O₂ or H₂O₂ plus JNK inhibitor (JNKi). SIRT1, acetylated histone H3 (Ac-H3) or total histone H3 were examined by Western blotting. C, Mt-SIRT1 shows similar activity to WT SIRT1 in deacetylation of p53. Experiment performed as in B, except that acetylated p53 (Ac-p53) and total p53 levels were monitored. The ratio Ac/Total shows the quantification of the expression levels of the acetylated substrates vs. the total levels of the same substrates. D, Mt-SIRT1 shows decreased activity in deacetylation of histone3 (H3). Cells were transfected with the empty vector (CMV), WT hSIRT1 (WT) or Mt-SIRT1 (Mt) then treated with anisomycin or anisomycin plus JNK inhibitor (JNKi). SIRT1, acetylated histone H3 or total H3 were examined by Western blotting. E, Mt-SIRT1 shows similar activity to WT SIRT1 in deacetylation of p53 when cells were treated with anisomycin. The experiment was performed as in D, and the levels of acetylated p53 (Ac-p53) and total p53 were determined by Western blotting.