Using a STAT3 antibody in chromatin immunoprecipitation (ChIP)

Fri, 10/21/2016 - 14:38

Signal transducer and activator of transcription 3 (STAT3) is an important oncogenic transcriptional factor that mediates tumor induced immune suppression.  Specifically, STAT3 transmits signals from cytokines and growth factor receptors in the plasma membrane (PM) to the nucleus, where they alter gene transcription.  Because of this transcriptional regulatory role, STAT3 also plays a part in regulating transcription of many critical genes that are involved in apoptosis, cell differentiation, immune response, tumor formation and more.  Using a STAT3 antibody in the application of chromatin immunoprecipitation (ChIP) is a very effective way to examine the relationship between genes and proteins in different molecular pathways and disease pathologies.  The next two studies used a STAT3 antibody in ChIP to investigate cell differentiation and lung tumor immune response in spermatogonia and granulocytes, respectively.   

STAT3 antibody

Jurkat human acute T cell leukemia cell line treated with 1000 U/mL Recombinant Human IFN‑ alpha Universal Type I (Catalog # 11200-1) for 1 hour was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. STAT3/DNA complexes were immunoprecipitated using 5 μg Goat Anti-Human/Mouse/Rat STAT3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1799) or control antibody (Catalog # AB‑108‑C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (Catalog # BAF109). Immunocomplexes were captured using 50 μL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. The c-myc promoter was detected by standard PCR.

Kaucher et al used a STAT3 antibody to find that NEUROG3 is activated downstream of STAT3 regulated progenitor cell spermatogonia differentiation during spermatogenesis.  First, it was established that inhibition of STAT3 in THY1+ spermatogonia resulted in reduced NEUROG3 expression.  Next, in order to determine whether STAT3 and NEUROG3 were part of the pathway that controls differentiation of spermatogonia, GDNF signaling was introduced to the THY1+ spermatogonia.  The introduction of GDNF to the THY1+ culture medium resulted in dephosphorylation of STAT3 and subsequent inhibition of NEUROG3.  With these findings, Kaucher et al used a STAT3 antibody in a ChIP experiment to understand exactly how STAT3 regulates gene expression on the THY1+ spermatogonia.   After an analysis on the nucleotide sequence of the promoter region of NEUROG3, a STAT3 binding site was discovered.  Using a STAT3 antibody, STAT3-DNA complexes were immunoprecipitated from THY1+ spermatogonia, and the ChIP analysis showed that the level of NEUROG3 binding by STAT3 correlated with treatments that alter the amount of NEUROG3.  Overall, this application helped to explain how STAT3 up regulates the expression of NEUROG3 and subsequent spermatogonia cellular differentiation.

Furthermore, Kreisel et al used a STAT3 antibody to investigate how Bcl3 regulates the emergency granulocyte response in an inflammatory lung injury.  After it was established that granulocyte signaling enhances Bcl3 expression, Kreisel et al set out to take a closer look at exactly how Bcl3 expression was regulated.  Using a ChIP assay with a STAT3 antibody on lineage depleted B6 bone marrow cells that were stimulated with G-CSF, IL-6 or IL-3.  These results yielded that STAT3 binds an enhanced promoter region of STAT3 following exposure to G-CSF or IL-6, but not IL-3. 

Novus Biologicals offers STAT3 reagents for your research needs including:

PMID: 22378757
PMID: 21157041
PMID: 20517723
PMID: 21441118

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