Validation of cloned Stat5-bound chromatin fragments from PRL-stimulated T-47D breast cancer cells. A. PRL-activated Stat5 binds in vitro to 7 of 10 (8 shown) Cloned Chromatin Fragments (CCF) obtained from a Stat5-chromatin library as shown by EMSA. Nuclear extracts from confluent T-47D cells overexpressing Stat5 and incubated with (+) or without (-) 10 nM human PRL for 30 min were used. Prolactin-induced protein-DNA complexes are indicated by the retarded migration of probe in the (+) PRL lanes for each of the CCFs. Supershift reactions, or band depletions, are indicated by the antibody (Ab) added and signify a specific Stat5-DNA interaction when compared to the supershift control, non-specific, purified IgG. B. Validation of in vivo Stat5 binding to immunocaptured Cloned Chromatin Fragments (CCFs) by ChIP assay. T-47D cells were incubated with (+) or without (-) PRL as described and Stat5-DNA complexes were enriched by chromatin immunoprecipitation. PCR primers specific for each CCF were designed and used for amplification of two independently generated pools of Stat5-chromatin interaction sites and PCR was performed twice on each pool; a representative experiment is shown. CCFs #5, #18, #23, #28, #29, and #30 were consistently positive for in vivo Stat5-DNA binding (upper panel). Positive PCR controls were performed on pre-IP template (lower panel).