Specificity of immunocapture of Stat5-bound chromatin in T-47D human breast cancer cells. A. Optimization of sonication parameters for shearing genomic DNA in T-47D cells. Sonication of formaldehyde-fixed cells for two 30 s pulses yielded ~400 bp chromatin fragments. The number and time of sonication pulses are listed above each lane. B. Prolactin (PRL) pretreatment did not affect chromatin fragmentation. T-47D cells were treated with (+) or without (-) 10 nM PRL for 30 min, fixed, sonicated (2 × 30 s), and a portion of the pre-IP samples were separated by agarose gel electrophoresis. C-E. Validation of quality of immunocaptured, Stat5-bound chromatin pool by ChIP analysis of known Stat5 target genes. Cells were incubated with (+) or without (-) 10 nM PRL for 30 min, and processed as described in Methods. Non-specific IgG was used as a negative IP control and PCR was performed using primers designed to specifically amplify the known Stat5 response element. PCR products shown are representative of at least two separate amplifications of at least two independently generated pools of Stat5-chromatin complexes. (-) PCR ctrl indicates a negative (no template) PCR control, (+) PCR ctrl Pre-IP indicates a positive PCR control performed on the pre-immunoprecipitated (input) DNA, and (+) PCR ctrl Gen indicates a positive PCR control performed on purified T-47D genomic DNA. PRL activated Stat5 specifically associated with the promoter of CISH (C) and β-Casein (D – upper panel), but not α2-Macroglobulin (D – lower panel), unless cells had been pretreated for four days with glucocorticoid (E – 1 μM dexamethasone; Dex).