(A) Generation of a library encoding STAT5-responsive genomic elements by ChIP-cloning. IL-2 stimulated, formaldehyde cross-linked YT cells were lysed, sonicated and immuno-precipitated with antibodies to STAT5A or STAT5B. Eluted DNA was ligated to a unidirectional linker (black blocks), amplified and then cloned into pCR II-TOPO vector. Clones containing inserts were identified by sequencing. (B) Successful immuno-precipitation of STAT5 from formaldehyde fixed YT cell lysates. YT cells were stimulated with medium (-) or IL-2 (+) then fixed with formaldehyde. Fixed lysates were immuno-precipitated with antibodies to STAT5 as indicated or normal rabbit serum (IgG CTRL) then Western blotted for STAT5. Molecular weight markers are indicated to the left side of the panel. Input material corresponds to 1% of cell lysate used in the immuno-precipitations. (C) Validation of STAT5 ChIP in YT cells. ChIP assay with C-terminal antibodies to STAT5A and B in combination (αSTAT5 C-term) or IgG control was carried out as described above. The eluted DNA was then used as template in qPCR reactions with primers designed to PRR III. (D) STAT5 bound genomic library captured by ChIP-cloning. Inserts were amplified via PCR using M13 primers prior to sequencing and visualized by agarose gel electrophoresis (1%). Stars (*) indicate clones without an insert. (E) Nearby gene mapping of the ChIP-clone identified genomic sequences. One hundred and nineteen clones were sequenced, 3 fragments were duplicates and 9 were greater than 300 kb away from any coding region. The remaining sequences that fell within 300 kb from coding regions were analyzed with Cis-Regulatory Element Annotation System (CEAS). The pie chart represents "%" distribution.