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. 2005 Aug 2;102(32):11278–11283. doi: 10.1073/pnas.0502738102

Fig. 4.

Fig. 4.

The association of Foxo1 and its cognate DNA sequence inhibits the PKB-dependent phosphorylation of Foxo1 in vitro. (A) In vitro kinase assays were performed with 1 μg of GST-Foxo1 (amino acids 157–268), 10 ng of PKB, and 0.5 mM ATP. (B) One microgram of wild-type GST-Foxo1 (amino acids 157–268) was preincubated with the indicated double-stranded oligonucleotides (0.5 or 1 pmol) and phosphorylated by 10 ng of PKB and 0.5 mM ATP in vitro. (C) Wild-type or mutated GST-Foxo1 (1 μg) was phosphorylated by PKB (0, 2, or 10 ng). (D) Wild-type or mutated GST-Foxo1 (1 μg) was preincubated with double-stranded oligonucleotide (0, 0.5, or 1 pmol) and phosphorylated by 10 ng of PKB. All reaction products were analyzed by Western blotting using anti-phospho-Foxo1 antibody or silver stain.