Transcriptional Regulation of LKB1 Figure 4. Binding of NF-Y transcription factor to CCAAT boxes within the LKB1 promoter. 32P-labeled double-stranded oligonucleotides harbouring the CCAAT boxes (their positions relative to the transcriptional start site, TSS, are indicated) (A) were incubated with 2 mg of nuclear extracts from ‘‘444’’ cells (lanes 1, 7 and 13) or from HepG2 cells (lanes 2–6, 8–12, and 14–18) and separated in a 7% polyacrylamide gel (B). Formation of sequence specific protein complexes was confirmed by competition with unlabeled oligos. Specific bands (indicated by arrows B, C and D) were competed by a 500-fold molar excess of wild-type oligo (WT; lanes 3, 9 and 15) but not by the same excess of mutant oligo (MUT; lanes 4, 10 and 16, respectively). Protein complexes containing the transcription factor NF-Y (arrow A) were further retarded by addition of an antibody against NF-Ya (NFY; lanes 5, 11 and 17) but not by the addition of the same amount of normal goat IgG (IgG; lanes 6, 12 and 18). doi:10.1371/journal.pone.0032590.g004 were performed. Indeed, a polyclonal antibody directed against the alpha subunit of NF-Y altered the mobility of the uppermost complex (‘‘complex A’’; lanes 5, 11 and 17, respectively) in comparison to the addition of a non-specific antibody (lanes 6, 12 and 18). Notably, the HepG2 specific ‘‘complex C’’ was not shifted by the NF-YA antibody, indicating that an additional protein, which is not present in ‘‘444’’ cells can bind to this element. Due to a different metabolic state, it is possible that the HepG2 specific complex may be necessary for the high transcriptional steady-state level of the LKB1 gene in liver cells [3]. Interaction between FOXO proteins and the LKB1 promoter Since mutation of a potential forkhead box transcription factor binding site within the LKB1 promoter significantly reduced promoter activity (Figure 1D), we assumed that FOXO proteins PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e32590