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. 2011 Sep;52(9):1693–1701. doi: 10.1194/jlr.M014647

Fig. 4.

Fig. 4.

Knockdown of SIRT1 leads to acetylation and inhibition of FoxO1 in adipocytes. A: Control (EV) and SIRT1-ablated (Sh) 3T3-L1 adipocytes on day 10 of differentiation were serum starved for 4 h, and FoxO1 was immunoprecipitated from the whole-cell lysates (500 µg). Proteins were eluted from the beads, and acetylated FoxO1 was analyzed by Western blotting with an antibody against acetylated lysine (Ac-Lys). Input lane shows 50 µg of the total cell lysates. B: Left panel, control (EV) and SIRT1-ablated (Sh) 3T3-L1 adipocytes on day 10 of differentiation were serum starved for 2 h, and total cell lysates (50 µg) were analyzed by Western blotting with the indicated antibodies. Right panel, relative band intensities for phosphorylated FoxO1 in EV (black bar) and Sh (white bar) cells were determined in three independent experiments and normalized by total FoxO1. Data are expressed as mean ± SD relative to the EV cells. * P < 0.05. C: Levels of mRNA in control (EV, black bars) and SIRT1-ablated (Sh, white bars) adipocytes were determined in triplicate by quantitative PCR and normalized by GAPDH mRNA levels. Data are expressed as mean ± SD relative to the expression levels in EV cells. 4E-BP1, eIF4E binding protein 1; SOD2, superoxide dismutase 2; PCK1, phosphoenolpyruvate carboxykinase 1. * P < 0.05; ** P < 0.001.