Anti foxo1 antibody

These were performed as previously described. Briefly, the cells were cross-linked with 1% (w/v) formaldehyde-PBS solutions for 10 min at room temperature, formaldehyde was then inactivated by the addition of 125 mM glycine. Chromatin extracts were sonicated to obtain DNA fragments with size of 300-800 bp and then they were immunoprecipitated overnight with rotation using the anti-p53 antibody (DO1,Santacruz Biotechnology) or the anti-Foxo1 antibody (Santa Cruz Biotechnology). On the following day, protein A/G magnetic beads(Millipore) that had been previously blocked with salmon sperm DNA were added to each reaction to precipitate antibody-DNA-protein complexes. The precipitated complexes were then separated using magnetic separator to separate immuno-precipitated complex and supernatant. The immuno-precipitated complex was then washed and incubated at 62°C overnight in parallel with ‘input’ samples to reverse the crosslinking of DNA. The DNA was then separated from the complex using a magnetic separator (Invitrogen), the DNA was purified using Qiagen-PCR purification kit prior to its use in the PCR reaction. For the immuno-depletion experiments the soup obtained after the ChIP with the first Ab, was incubated with anti-FOXO-1 or anti-p53 antibodies and subjected to a second ChIP. The precipitated DNA was subjected to PCR reactions for 30–35 cycles as previously described.

A polyclonal-specific antibody against HMGA1^{23 (link)} was used to analyze HMGA1 protein expression in HEK-293 and HepG2 cells, and in liver nuclear extracts from normal and Hmga1 mutant mice. An anti-FoxO1 antibody (Santa Cruz Biotechnology) was employed to measure total FoxO1 in both human cell lines and animal tissues. An anti phospho-FoxO1 (Thr24) (Cell Signaling Technology) was used to analyze the levels of pFoxO1 protein in both nuclear and cytoplasmatic extracts.