FIGURE 6.

FoxO3a binds to bim promoter to activate transcription. A, nucleotide sequence of the 442-bp fragment of the porcine bim promoter aligned to the corresponding mouse sequence. Identical bases are indicated by asterisks, and amino acids with conserved similarities are indicated by dots or colons. The arrow indicates the first nucleotide of the transcription start site. The putative Forkhead-responsive element is boxed. B, granulosa cells in Lysis Buffer at a cell concentration of 1 × 10⁷ per ml sheared with six sets of 10-s pulses (40 watts) on wet ice using a high intensity ultrasonic sonicator (Cole Parmer). Sheared chromatin was then electrophoresed through a 2% agarose gel and stained with ethidium bromide. The picture shows that the majority of the DNA has been sheared to a length between 200 and 1000 bp. C, chromatin fragments were subjected to immunoprecipitation with antibodies to IgG (nonspecific) or GFP as indicated. Upon DNA isolation, amplification of the bim promoter DNA was assessed by PCR and gel electrophoresis. Input DNA was used as positive control. A representative graph of three independent experiments is shown.