c-Jun Activation Kit

• Quantify endogenous c-Jun activation specifically and reliably in whole cells
• Immunofluorescence-based screening assay provides sensitive quantitative and qualitativeresults without radioactivity
• No cell lysis, purification, or filtration steps
• No need to generate stable clonal lines; entire assay completed in 3 h (post-stimulation)
• Earlier and more definitive measure of transcription factor activation relative to electophoretic mobility shift or gene reporter assay

The c-Jun Activation kit provides the reagents and protocol necessary to quantify c-Jun activation by directly measuring its translocation from the cytoplasm to the nucleus. The assay is performed on live cells growing on standard high-density microplates or microscope slides. The kit contains an anti-phospho-c-Jun primary antibody and a DyLight™ 488-Conjugated Secondary Antibody. The nuclear region is identified by Hoechst Dye, also included in the kit.

c-Jun is a transcription factor that is activated through phosphorylation in the activation domain by Jun-N terminal kinases (JNKs). Phosphorylated Jun family members then form homodimers or heterodimeric complexes with Fos, composing the AP-1 transcription factor, which migrates into the nucleus. The AP-1 transcription factor has been implicated in stress-induced apoptosis, heat shock, T-cell activation, cellular responses to inflammatory cytokines, and cellular transformation, as well as ischemia/reperfusion. c-Jun is activated by a wide variety of stimuli, including anisomycin, epidermal growth factor, hydrogen peroxide and TNFα.

The c-Jun Activation HCS Reagent Kit, in combination with the Thermo Scientific ArrayScan® HCS Reader and the Cytoplasm to Nucleus Translocation BioApplication software enable automated plate handling, focusing, cell image acquisition, analysis and quantification of c-Jun activation. For a more detailed description of the image processing algorithm, see the Cytoplasm to Nucleus Translocation BioApplication Guide that accompanies the Cytoplasm to Nucleus Translocation BioApplication Software.

Figure 1. Images of fluorescently labeled HeLa cells before and after activation of c-Jun by anisomycin (500 ng/mL for 25 min). The left panel shows c-Jun localization in unstimulated cells. Nuclei appear blue due to low levels of nuclear (phosphorylated) c-Jun. The right panel shows c-Jun localization in stimulated cells. Blue-green color of nuclei is due to colocalization.

Figure 2. Diagrammatic view of the principle of the c-Jun activation. c-Jun (green), normally present in the cytoplasm, translocates to the nucleus upon stimulation.

Figure 3.  Dose response curve for c-Jun activation by anisomycin in HeLa cells.

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