ID_REF = VALUE = Expression values were converted from log 2.
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
The raw data in the .CEL files were normalized by the GCRMA method (GC robust multi-array analysis). It is implemented in Bioconductor (http://www.bioconductor.org/). The raw signal intensity data were normalized, background corrected and summarized based on certain statistical models, and an expression value, in log2-scale, is obtained per chip per probe set.
Hybridization, etc was performed by the BCM Microarray Core Facility in Houston, TX.
Biotinylated cRNA were prepared according to the standard Affymetrix protocol in the Baylor College of Medicine Microarray Core Facility, Houston, TX.
Arrays were scanned on an Affymetrix Scanner 3000 7G
Data was analyzed using MAT at http://liulab.dfci.harvard.edu/MAT/ CplusInp.bed.txt contains all 1581 high quality STAT5 binding sites.
Approximately 8.25μg of DNA was hybridzed per array using the Affymetrix hybridization kit at the EMBL Genomics Core Facility in Heidelberg, Germany.
To generate libraries from ChIP-ed DNA, random amplification was carried out as described at http://research.stowers-institute.org/gertonlab/protocols/RandomPCRamplification.pdf by the DeRisi lab at the UC San Francisco.
Cells were maintained in RPMI-1640 medium containing 10% fetal calf serum, 2 mM L-glutamine and penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively). Kit225 media was supplemented with 20 U/ml human recombinant IL-2 (NCI Preclinical Repository).
Prior to IL-2 stimulation, Kit225 cells were made quiescent for 24 h in their regular medium without IL-2. Cell stimulations were carried out with 10 nM IL-2.
Extraction of total RNA was performed using Qiagen Rneasy Kit according to the manufacturer's instructions.
Chromatin immunoprecipitations were performed from approximately 5 x 107 Kit225 cells as described by our group earlier (Nagy et al Mol. Cancer. 2009) with anti-STAT5A/B antibodies (mixing sc-1081 and sc-835 (C-terminal STAT5A and STAT5B antibodies, respectively)) or normal rabbit serum (IgG control) for 3h at 4 ºC. To confirm that the assays were successful, PCR amplification of the known STAT5 binding site located 5’ to the human IL2RA gene within the Positive Regulatory Region III (Forward: 5’-ACG TCT AGA AAG AAA GTG GTC-3’ Reverse: 5’- CTG TCC CTG GAT GAA CCT AGT-3’) was performed using quantitative real time PCR.