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. 2018 Aug 28;46(18):9601–9616. doi: 10.1093/nar/gky777

Figure 1.

Figure 1.

NAT10 is deacetylated by Sirt1 upon energy deprivation. (A) HCT116 cells were transfected with the indicated plasmids. Cell lysates were prepared and subjected to immunoprecipitation using an anti-acetyl-lysine antibody. The acetylation levels of NAT10 in the immunoprecipitants were evaluated by western blot using an anti-NAT10 antibody. (B) The indicated plasmids were coexpressed in HCT116 cells. Cell lysates were prepared and subjected to immunoprecipitation using an anti-acetyl-lysine antibody. The acetylation levels of NAT10 were evaluated by Western blot using an anti-NAT10 antibody. (C) Purified His-NAT10 was preacetylated and incubated with purified Flag-Sirt1 in the absence or presence of 1 mM NAD+. The acetylation levels of NAT10 were evaluated by Western blot with an anti-acetyl-lysine antibody. (D) Flag-Sirt1 was transfected into HCT116 cells. Immunoprecipitation was performed with an anti-Flag antibody, and NAT10 expression in the precipitants was determined by Western blot. (E) Flag-NAT10 was transfected into HCT116 cells. Immunoprecipitation was performed with an anti-Flag antibody, and Sirt1 expression in the precipitants was determined by Western blot. (F) A GST pull-down experiment was performed with GST-Sirt1 and purified Flag-NAT10. The binding of GST-Sirt1 to Flag-NAT10 was detected by immunoblotting with an anti-Flag antibody. (G) A GST pull-down experiment was performed using purified GST-NAT10 deletion mutants and purified Flag-Sirt1. The schematic diagram represents the GST-NAT10 deletion mutant constructs (lower panel). (H) HCT116 cells were cultured in medium containing 1.0 g/l, 0.5 g/l, or 0 g/l glucose for 18 h. The cells were harvested, and immunoprecipitation was performed using an anti-acetyl-lysine antibody. The acetylation levels of NAT10 were evaluated by Western blot using an anti-NAT10 antibody. (I) HCT116 cells were cultured in medium containing 1.0 g/l, 0.5 g/l, or 0 g/l glucose for 18 h. Immunoprecipitation was performed with an anti-Sirt1 antibody, and NAT10 expression in the precipitants was evaluated by immunoblotting. (J) HCT116 cells were transfected with the indicated siRNAs. Cells were cultured in glucose-rich or glucose-free medium for 18 h, harvested and then subjected to immunoprecipitation with an anti-acetyl-lysine antibody. The acetylation levels of NAT10 were evaluated by Western blot using an anti-NAT10 antibody on the immunoprecipitants. See also Supplementary Figure S1.