Chromatin immunoprecipitation analysis of Phospho-STAT5 alpha (pTyr694) was performed using cross-linked chromatin from 1 x 10⁶ HCT 116 (Human colorectal carcinoma cell line) cells treated with serum for 0, 15, and 30 minutes.

Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 μL/100 μL well volume of ab277762. Chromatin aliquots from ~1 x 10⁵ cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 μL of eluted DNA in 2 μL SYBR real-time PCR reactions containing primers to amplify exon-1 or exon-2 of human Egr-1, or the imprinting control region (ICR) of the human H19 locus. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments.

A schematic representation of the human Egr-1 and H19 loci are shown above the data where boxes represent exons (black boxes = translated regions, white boxes = UTRs), the zigzag lines represent introns, and the straight line represents upstream sequence. Regions amplified by Egr-1 and H19 primers are represented by black bars.

Data courtesy of the Innovators Program.

Immunohistochemistry analysis of STAT5 Alpha (pY694) showing staining in the cytoplasm and nucleus of paraffin-embedded human diffuse large B cell lymphoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H₂O₂-methanol for 15 min at room temperature, washed with ddH₂O and PBS, and then probed with ab277762 diluted in 3% BSA-PBS at 1/20 dilution overnight at +4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

All lanes : Anti-STAT5a (phospho Y694) antibody [6HCLC] (ab277762) at 2.5 µg/ml

Lane 1 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : Preincubation with the phosphopeptide, Jurkat whole cell lysate with Phosphopeptide

Predicted band size: 90 kDa

Enrichment of endogenous STAT5 alpha pTyr694 protein at specific gene loci using ab277762:

Chromatin Immunoprecipitation (ChIP) was performed using Aab277762 (5 μg) on sheared chromatin from 2 million HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with 100 ng/mL of IFN gamma for 45 minutes using the "MAGnify ChIP system" kit. Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system with optimized PCR primer pairs for the promoter of active COX1, AP1 gene, used as positive control target, and the SAT2, used as negative control target. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Immunohistochemistry analysis of STAT5 Alpha (pY694) showing staining in the cytoplasm and nucleus of paraffin-embedded human lung squamous carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H₂O₂-methanol for 15 min at room temperature, washed with ddH₂O and PBS, and then probed with ab277762 diluted in 3% BSA-PBS at a dilution of 1/20 overnight at +4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.