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. 2011 Dec 7;287(6):3850–3858. doi: 10.1074/jbc.M111.317404

FIGURE 2.

FIGURE 2.

SIRT1 specifically deacetylates PGAM1. A, only SIRT1 knockdown significantly increased PGAM1-acetylated levels. HEK293 cells were transiently transfected with MYC-PGAM1 and siRNA to SIRT1–3. Soluble extracts were resolved by SDS-PAGE and detected through Western blot (WB), with anti-acetyl-lysine (AcK) and anti-PGAM1 antibodies. Input blots (1% total cellular extract) were detected with antibodies against PGAM1, SIRT1, SIRT2, SIRT3, and tubulin. B and C, activity was measured at 340 nm by PGAM1-coupled assay. D, acetylation of PGAM1 is decreased in vitro by SIRT1 deacetylation. Immunoprecipitants (IP) of MYC-PGAM1 from HEK293 cells transiently transfected with PGAM1 were incubated with the combination of NAD+ and SIRT1, and the reactions were resolved by SDS-PAGE and detected through Western blot, with anti-acetyl-lysine and anti-PGAM1 antibodies. E, activity was measured at 340 nm by PGAM1-coupled assay. Error bars represent S.E. (n = 3); *, p < 0.05 compared with control.