The protocol is performed on live cells growing on standard high-density microplates. The kit is supplied with a primary STAT2 antibody and a DyLight™ 488-conjugated Secondary Antibody. The nuclear region is identified by Hoechst Dye, which is also included.
Cytokines are important for proliferation and differentiation of hematopoietic cells. Many cells respond to cytokine induction via the janus kinases-signal transducers and activators of transcription (JAK/STAT) pathway, resulting in the transcription of selected genes. The complement of induced genes and associated response varies according to the cell type and stimulus, which is reflected by tyrosine phosphorylation by specific JAKs. Consequently, it is crucial that screens for potential drugs that affect this collection of pathways consider specificity among the six known STAT targets. This specificity is addressed by this kit because activation of STAT2 is quantified by its translocation to the nucleus.
The STAT2 Activation Kit enables screening of STAT2 inhibitors by stimulating cells with a control inducer such as interferonα (IFN-α) after exposing live cells to test compounds. Replacing IFN-α in the assay with test compounds identifies agonists of STAT2 translocation. Translocation is directly quantified as the difference in cytoplasmic to nuclear intensity of the labeled transcription factor. This kit, in combination with the Thermo Scientific ArrayScan HCS Reader and the Cytoplasm to Nucleus Translocation BioApplication software, enables automated plate handling, focusing, cell image acquisition, analysis and quantification of STAT2 activation.
Figure 1. Stained HeLa cells before and after activation of STAT2. Top panel: STAT2 localization in unstimulated cells. Bottom panel: STAT2 localization in cells stimulated with 500 U/ml of IFN-α for 20 minutes.
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