Fig. 2.

Recruitment of Stat5a to the β-casein promoter and distal enhancer in HC-11 cells is impaired by progesterone. HC-11 cells were transduced with 50 pfu/cell of adenovirus encoding hPR-B and then treated for 1 h with either: ethyl alcohol (EtOH) (−control), ovine PRL (3 μg/ml)/HC (1 μg/ml), R5020 (100 nm), or ovine PRL/HC + R5020 (same concentrations as given alone). Cells were then harvested, lysed, and processed for ChIP with antibodies to Stat5 (L-20) assay using PCR primers flanking the Stat5 response element (S5RE) and GRE half-sites within the proximal promoter of β-casein (−190 to −20) or to the distal enhancer region (−6400 to 6000). A, Schematic representation of β-casein proximal promoter and distal enhancer with forward (Fwd) and reverse (Rev) primer sets used for PCR denoted. B, Stat5a binding to the proximal promoter and distal enhancer in response to treatment with of PRL/HC for different time points between 5 min and 24 h. C, Stat5a binding to the proximal promoter and distal enhancer in the presence of PRL/HC and R5020 for time points between 0 and 24 h. Quantification of ChIP assays from multiple experiments was performed by agarose gel electrophoresis of PCR-amplified input and immunoprecipitated DNA and image scanning analysis of band intensitives with Syngene Genetools software. Data represent the average density of immunoprecipitated DNA as a percentage of input DNA on the agarose gels adjusted after subtracting signals obtained with control IgG. The values are averages ± sem from three independent experiments. Insets are representative agarose gels of PCR products from one of the three experiments. Oct, Octamer transcription factor; Ets, Ets transcription factor; NF-1, nuclear factor 1.