Phospho-ATF2 (Thr71) cellular kit
This HTRF kit enables both SAPK/JNK and p38 MAPK pathway readout, measuring ATF2 modulation phosphorylated at Threonine-71.
- Highly specific
ATF2 (Activating transcription factor 2) is a transcription factor activated by phosphorylation on threonin residues by JNK/SAPK. The phospho-ATF2 (Thr71) kit enables the quantitative detection of transcription factor ATF2 modulation phosphorylated at Threonine-71. ATF2 is also involved in PI3K/AKT, TNF, and estrogen signaling along with insulin secretion, thyroid hormone release, substance dependence, viral carcinogenesis and infectious diseases. The simple add-and-read protocol features no wash steps for a faster analysis in biological applications like oncology, diabetes or inflammation.
Mouse NIH3T3 cells were seeded at different cell densities in 96-well microplates, then stimulated with increasing concentrations of anisomycin for 30 minutes. Following the 2-plate assay protocol, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-ATF2 (Thr71) detection reagents. The HTRF signal was recorded after an overnight incubation.
Human HEK293A suspension cells were seeded at 100,000 cells/well in a 96 well half area plate, and incubated for 24h at 37°C, 5% CO2. Then cells were stimulated with different concentrations of anisomycin for 15, 30 & 60 minutes. After lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-ATF2 (Thr71) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
The human NIH3T3 cell line was seeded in a T175 flask and incubated a 37°C, 5% CO2 until confluency.
Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-ATF2 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.
Using the HTRF Phospho-ATF2 T71 assay, 3,000 cells/well were sufficient to detect a signal while 6,125 cells were needed using Western Blot with an ECL detection. These results demonstrate that the HTRF phospho-ATF2 assay is 2 times more sensitive than the Western Blot.
In response to cellular stress, such as exposure to genotoxic agents, cytokines, or UV irradiation, SAPK/JNK and p38 MAP kinases are activated and phosphorylate the transcription factor ATF-2 on residues Thr69 and Thr71. Once in the nucleus, phospho-ATF2 binds to cAMP response element (CRE) or to Activator Protein-1 (AP-1) response elements, and regulates the transcription of genes involved in cell growth, survival, and DNA damage response.
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