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. 2016 Feb 12;7:10734. doi: 10.1038/ncomms10734

Figure 3. SIRT7 interacts with and deacetylates U3-55k.

Figure 3

(a) Acetylation of U3-55k is increased on NAM treatment. HEK293T cells expressing Flag-U3-55k were treated for 6 h with 500 nM of TSA, 10 mM of NAM, or were left untreated (−). Acetylation of immunopurified Flag-U3-55k was analysed on western blots using anti-pan-AcK antibody. The blot was reprobed with anti-Flag antibody to monitor the amount of Flag-U3-55k. The numbers below indicate the relative level of acetylation. (b) PCAF acetylates U3-55k. HEK293T cells expressing GFP-U3-55k were transfected with siRNA against PCAF (siPCAF) or control siRNA (siCtrl). Acetylation of GFP-U3-55k purified from cells cultured in the absence or presence of NAM (10 mM, 6 h) was detected on western blots with anti-pan-AcK antibody; equal loading of GFP-U3-55k was confirmed by reprobing with anti-GFP (upper panels). Cell lysates were probed with anti-PCAF and anti-tubulin antibodies to demonstrate depletion of PCAF by siRNA (lower panels). (c) SIRT7 interacts with U3-55k. Lysates from HEK293T cells co-expressing Flag-U3-55k and GFP-tagged SIRT1, SIRT6 or SIRT7 were incubated with anti-Flag antibody and co-precipitated sirtuins were analysed on western blots with anti-GFP antibodies. (d) Association of endogenous SIRT7 with U3-55k. SIRT7 was immunoprecipitated with 5 μg of anti-SIRT7 antibodies and co-precipitated U3-55k was detected on western blots with anti-U3-55k antibodies. (e) Overexpression of GFP-SIRT7 decreases acetylation of U3-55k. Flag-U3-55k was immunopurified from HEK293T cells co-expressing GFP-SIRT7, GFP-SIRT1 or Flag-U3-55k alone (Ctrl). Western blots show the level of acetylated Flag-U3-55k probed with anti-pan-AcK antibody, purified Flag-U3-55k was detected with anti-Flag antibody. (f) Depletion of SIRT7 leads to hyperacetylation of U3-55k. Western blots monitoring acetylation of Flag-U3-55k from HEK293T cells treated with control or SIRT7-specific shRNA (left panels), or from parental HEK293T cells (SIRT7+/+) and SIRT7−/− cell lines sg2–8 and sg4–6 (right panel; see also Supplementary Fig. 4c). Membranes were probed with antibodies against pan-AcK, Flag, SIRT7 and actin. (g) SIRT7 deacetylates U3-55k in vitro. Reactions contained 0.8 μg of immunopurified Flag-SIRT7, 2 mM of NAD+, or 10 mM of NAM and 1 μg of bead-bound GFP-U3-55k (Supplementary Fig. 4e). Acetylation of GFP-U3-55k was detected with anti-pan-AcK antibody, GFP-U3-55k was analysed with anti-GFP antibody.