Fig 2: Kinetic characteristics of SIRT6 deacetylase activity.(A) Progress curves for purified, recombinant full-length wild-type (wt) and mutant SIRT6 proteins, expressed in E.coli. ? wt SIRT6 (2 µM), ?H133W (10 µM), ? H133Y (10 µM), ? N114A (10 µM), ? inhibition of wt (2 µM) with 20 mM NAM (this also generates an intrinsic level of background fluorescence). Error bars represent the standard error of mean duplicate values. (B) K m,app determination for peptide gave a value of 14.7+/-1.5 µM. Error bars represent the standard error of mean duplicate values. (C) K m,app determination for NAD+ gave a value of 52.4+/-18 µM. Error bars represent the standard error of mean duplicate values. (D) Deacetylation of purified H3/H4 histones with SIRT6. Deacetylation could be detected at a substrate:enzyme ratio of 1:1.

Fig 3: Interaction of SIRT6 with RelA/p65.HEK293 cells were transiently transfected with Flag-tagged SIRT6. Cell extracts were prepared and immunoprecipitated with an antibody to RelA/p65 and analysed by Western blotting (WB) with antibodies to RelA/p65 (lanes 1–4) SIRT6 (lane 5–8) and Flag (lane 9–12). Lanes 1,5 and 9 show the anti-RelA/p65 immunoprecipitate (p65 IP), lanes 2,6 and 10 show the non-immunoprecipitated or “flow through” material (FT), lanes 3,7 and 11 show a control immunoprecipitation using only Protein G agarose (p65 control) and lanes 4,8 and 12 are a sample of the total cell extract (input).

Fig 4: PIAS1 SIRT6 co-immunoprecipitation.HEK293 cell extracts were prepared and immunoprecipitated with an antibody to PIAS1 (lane 1 aPIAS1 IP) or an irrelevant antibody (lane 2 Control IP) followed by Western blotting with anti-PIAS1 antibody. The PIAS1 band was seen to migrate just above the 62 kDa marker. HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 (lane 3) or the H133W mutant (lane 4) and immunoprecipitated with anti-Flag followed by Western blotting with anti-PIAS1 (a-Flag IP). In a separate experiment HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 and cell extracts were prepared and immunoprecipitated with an anti-Flag antibody and analysed by Western blotting with anti-PIAS1 antibody (lanes 5–8). Lane 5 represents the total cell extract used in the immunoprecipitation (input). Lane 6 represents the anti-Flag immunoprecipitation eluted from the beads with Flag peptide (a-Flag IP). Lanes 7 represents the non-immunoprecipitated “flow through” material from the cell extract (FT) and lanes 8 represents the immunoprecipitated material eluted from the beads with SDS-PAGE sample buffer (a-Flag IP).

Fig 5: SMARCA5 SIRT6 co-immunoprecipitation.HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6. Cell extracts were prepared and immunoprecipitated with an irrelevant antibody (lane 1 Control IP) or an antibody to SMARCA5 (lane 2 αSMARCA5 IP). The SMARCA5 band was seen to migrate above the 98 kDa marker. In a separate experiment HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 and cell extracts were prepared and immunoprecipitated with an anti-Flag antibody and analysed by Western blotting with anti-SMARCA5 antibody (lanes 3–6). Lane 3 represents the total cell extract used in the immunoprecipitation (input). Lane 4 represents the anti-Flag immunoprecipitation eluted from the beads with Flag peptide (α-Flag IP). Lanes 5 represents the non-immunoprecipitated “flow through” material from the cell extract (FT) and lanes 6 represents the immunoprecipitated material eluted from the beads with SDS-PAGE sample buffer (α-Flag IP).