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. 2005 Oct 15;19(20):2435–2446. doi: 10.1101/gad.1340505

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Copyright © 2005, Cold Spring Harbor Laboratory Press

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Figure 7. — FOXO1 binds and activates InR promoter in the absence of insulin. (A) ChIP was performed in C2C12 cells incubated in serum-free medium with (1 μM) or without insulin. Antibodies against FOXO1 specifically precipitate InR promoter only in the absence of insulin. Actin was used to normalize the qPCR between samples, and each value was independently normalized to its input. (B) FOXO1 is dephosphorylated in the absence of insulin. (C) mRNA for InR is up-regulated in the absence of insulin. (D) FOXO1 (lane 1) and FOXO1A3 (lane 2) were phosphorylated in vitro by Akt kinase and detected by Western blot with anti-phospho-FOXO1-specific antibodies (upper panel) or FOXO1-specific antibodies (lower panel). (Lanes 3,4) Coomassie gel of both proteins. (E) Phosphorylation by Akt prevents binding of FOXO1 to the InR promoter probe. Band shift performed with recombinant human FOXO1 (shown in D) and a DNA probe containing the putative FRE from the InR promoter. (Lane 1) No protein. (Lanes 2-5) FOXO1. (Lanes 6-9) FOXO1A3. (Lanes 10,11) Band shift was performed with FOXO1 after (+) or before (-) in vitro kinase reaction.

Figure 7. — FOXO1 binds and activates InR promoter in the absence of insulin. (A) ChIP was performed in C2C12 cells incubated in serum-free medium with (1 μM) or without insulin. Antibodies against FOXO1 specifically precipitate InR promoter only in the absence of insulin. Actin was used to normalize the qPCR between samples, and each value was independently normalized to its input. (B) FOXO1 is dephosphorylated in the absence of insulin. (C) mRNA for InR is up-regulated in the absence of insulin. (D) FOXO1 (lane 1) and FOXO1A3 (lane 2) were phosphorylated in vitro by Akt kinase and detected by Western blot with anti-phospho-FOXO1-specific antibodies (upper panel) or FOXO1-specific antibodies (lower panel). (Lanes 3,4) Coomassie gel of both proteins. (E) Phosphorylation by Akt prevents binding of FOXO1 to the InR promoter probe. Band shift performed with recombinant human FOXO1 (shown in D) and a DNA probe containing the putative FRE from the InR promoter. (Lane 1) No protein. (Lanes 2-5) FOXO1. (Lanes 6-9) FOXO1A3. (Lanes 10,11) Band shift was performed with FOXO1 after (+) or before (-) in vitro kinase reaction.