Intracellular Staining by Flow Cytometry
|Detection of STAT5a in Jurkat Human Cell Line by Flow Cytometry. Jurkat human acute T cell leukemia cell line was stained with Mouse Anti-Human STAT5a Monoclonal Antibody (Catalog # MAB21741, filled histogram) or isotype control antibody (Catalog # MAB002, open histogram), followed by Allophycocyanin-conjugated Anti-Mouse IgG F(ab')2 Secondary Antibody (Catalog # F0101B). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with methanol.|
|STAT5a in K562 Human Cell Line. STAT5a was detected in immersion fixed K562 human chronic myelogenous leukemia cell line using Mouse Anti-Human STAT5a Monoclonal Antibody (Catalog # MAB21741) at 25 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.|
STAT5a (Signal Transducer and Activator of Transcription-5a) is one of two closely related genes that belong to the STAT family of transcription factors. It is a 91 kDa cytosolic protein that contains an N-terminal domain (with an NES/Nuclear Export Signal) a coiled-coiled region (with an NLS/Nuclear Localization Signal) a DNA-binding site that recognizes a GAS (Gamma-interferon Activated Site) motif, an SH2 domain that allows for dimerization, and a C-terminal transactivation domain. STAT5a likely exists in a quiescent state as a cytoplasmic anti-parallel homodimer. Following activation of both tyrosine and non-tyrosine kinase membrane-bound receptors, STAT5a is phosphorylated, increasing its MW by some 5-6 kDa. Phosphorylated STAT5a will enter the nucleus as either a homodimer (or heterodimer with STAT5b), or as a complex with the intracellular domain of a tyrosine kinase receptor such as ErbB4. Once in the nucleus, STAT5a will form a homotetramer and bind either GAS or GAS-related sequences in gene regulatory regions. Notably, it is suggested that STAT5a may "cycle" through the nucleus without phosphorylation, a process that would seem to involve Importins alpha 3 and beta 1. STAT5a is related to STAT5b through gene duplication. They show 92% amino acid (aa) sequence identity, with the major differences existing over aa 45-56 and 773-794. The genes are not entirely redundant; STAT5a activates NDGR1 and SH2, while STAT5b regulates the Treg T cell genes, FoxP3 and CD25/IL-2R alpha.
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