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. 2014 Oct 2;5(10):e1432. doi: 10.1038/cddis.2014.406

Figure 8.

Figure 8

Acetylated HMGB1 is an effective substrate for SIRT1. (a) HEK293T cells were transfected with empty vector (pcDNA3.1), pcDNA3.1-Flag-HMGB1, or pcDNA3.1-Myc-SIRT1. (b) HEK293T cells were transfected with pcDNA3.1-HA-PCAF or increasing amounts (2 μg and 4 μg) of pcDNA3.1-Myc-SIRT1. After incubation for 48 h, the cells were stimulated with (a) or without (b) LPS for 3 h. (c) Whole-cell lysates from SIRT1-knockout (SIRT1−/−) or wild-type (SIRT1+/+) MEFs were pulled down with anti-HMGB1 and analyzed by western blotting with anti-acetyl-lysine, anti-HMGB1, or anti-SIRT1 antibody to detect acetylated HMGB1 or total HMGB1 and SIRT1. (d) SIRT1−/− MEFs were transfected with empty vector (pcDNA3.1-Myc) or pcDNA3.1-Myc-SIRT1 and incubated for 48 h, after which the cells were harvested and subjected to immunoprecipitation with anti-HMGB1 antibody. Acetylated HMGB1 or total HMGB1 and SIRT1 were detected by western blot analysis. β-actin was used as a loading control