Figure 1: Treatment of NIH/3T3 cells with anisomycin stimulates phosphorylation of ATF-2 at Thr71, detected by PathScan® Phospho-ATF-2 (Thr71) Sandwich ELISA kit #7185, but does not affect the level of total ATF-2 protein detected by PathScan® Total ATF-2 ELISA kit #7195. OD 450 readings are shown in the top portion of the figure, while the corresponding Western blot using ATF-2 Rabbit mAb #9226 (left panel) or phospho-ATF-2 (Thr71) Antibody #9221 (right panel) is shown in the bottom portion of the figure.
Figure 2: Relationship between protein concentration of lysates from untreated and anisomycin-treated NIH/3T3 cells and kit assay optical density readings. NIH/3T3 cells (80% confluence) were treated with anisomycin and lysed after incubation at 37ºC for 30 minutes.
CST's PathScan® Phospho-ATF-2 (Thr71) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-ATF-2 (Thr71) protein. A Phospho-ATF-2 (Thr 71) Antibody has been coated onto the microwells. After incubation with cell lysates, phospho-ATF-2 protein is captured by the coated antibody. Following extensive washing, a total ATF-2 Mouse mAb is added to detect the captured phospho-ATF-2 protein. HRP-linked Anti-mouse Antibody #7076 is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-ATF-2 (Thr71).
Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Phospho-ATF-2 (Thr71) Sandwich ELISA Kit detects endogenous levels of Phospho-ATF-2 protein. Using this Sandwich ELISA Kit #7185, a significant induction of phospho-ATF-2 in NIH/3T3 cells treated with anisomycin can be detected. However, the level of total ATF-2 (phospho- and nonphospho-), detected by PathScan® Total ATF-2 Sandwich ELISA Kit #7195, remains unchanged (Figure 1). This kit can also be used to detect phosphorylated ATF-2 protein in human HeLa and 293 cells. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
The transcription factor ATF-2 (also called CRE-BP1) binds to both AP-1 and CRE DNA response elements and is a member of the ATF/CREB family of leucine zipper proteins (1). ATF-2 interacts with a variety of viral oncoproteins and cellular tumor suppressors and is a target of the SAPK/JNK and p38 MAP kinase signaling pathways (2-4). Various forms of cellular stress, including genotoxic agents, inflammatory cytokines, and UV irradiation, stimulate the transcriptional activity of ATF-2. Cellular stress activates ATF-2 by phosphorylation of Thr69 and Thr71 (2-4). Both SAPK and p38 MAPK have been shown to phosphorylate ATF-2 at these sites in vitro and in cells transfected with ATF-2. Mutations of these sites result in the loss of stress-induced transcription by ATF-2 (2-4). In addition, mutations at these sites reduce the ability of E1A and Rb to stimulate gene expression via ATF-2 (2).
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