Figure 1: Treatment of 293 cells with UV stimulates phosphorylation of c-Jun at Ser63, detected by PathScan® Phospho-c-Jun (Ser63) Sandwich ELISA Kit II #7145, but does not affect the level of total c-Jun protein detected by PathScan® Total c-Jun Sandwich ELISA Kit II #7150. λ protein phosphatase (LPP) treatment of control cell lysates (37ºC for 90 minutes) abolished the basal phosphorylation of c-Jun in control lysates shown in both Sandwich ELISA and western analysis. The OD450 readings are shown in the top figure, while the corresponding western blot, using Phospho-c-Jun (Ser63) rabbit mAb #2378 (right panel) or c-Jun rabbit mAb (6H5) #2374 (left panel), is shown in the bottom figure.
Figure 2: Linear relationship between protein concentration of lysates from UV-treated 293 cells and kit assay optical density readings. Pathscan® Total c-Jun Sandwich ELISA Kit II #7150 is more sensitive than kit #7270 over a range of lysate concentrations. 293 cells (70-90% confluence) were treated without or with UV and lysed after incubation at 37ºC for 30 minutes.
CST's PathScan® Total c-Jun Sandwich ELISA Kit II is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total c-Jun protein. A c-Jun rabbit mAb has been coated onto the microwells. After incubation with cell lysates, c-Jun protein is captured by the coated antibody. Following extensive washing, c-Jun Mouse mAb is added to detect the captured c-Jun protein. Anti-mouse IgG, HRP-linked antibody 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 total c-Jun protein.
Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Total c-Jun Sandwich ELISA Kit II detects endogenous levels of total c-Jun protein. As shown in Figure 1, using PathScan® Phospho-c-Jun (Ser63) Sandwich ELISA Kit II #7145, a significant induction of phospho-c-Jun (Ser63) in UV-treated 293 cells can be detected. However, the level of total c-Jun protein (phospho and non-phospho), detected by this Sandwich ELISA Kit #7150, remains unchanged. Both C6 and NIH/3T3 cells treated with UV light or anisomycin show similar results (data not shown).
This kit is more sensitive than kit #7270 (figure 2). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
c-Jun is a member of the Jun family containing c-Jun, JunB, and JunD, and is a component of the transcription factor activator protein-1 (AP-1). AP-1 is composed of dimers of Fos, Jun, and ATF family members and binds to and activates transcription at TRE/AP-1 elements (reviewed in 1). Extracellular signals including growth factors, chemokines, and stress activate AP-1-dependent transcription. The transcriptional activity of c-Jun is regulated by phosphorylation at Ser63 and Ser73 through SAPK/JNK (reviewed in 2). Knock-out studies in mice have shown that c-Jun is essential for embryogenesis (3), and subsequent studies have demonstrated roles for c-Jun in various tissues and developmental processes including axon regeneration (4), liver regeneration (5), and T cell development (6). AP-1 regulated genes exert diverse biological functions including cell proliferation, differentiation, and apoptosis, as well as transformation, invasion and metastasis, depending on cell type and context (7-9). Other target genes regulate survival, as well as hypoxia and angiogenesis (8,10). Research studies have implicated c-Jun as a promising therapeutic target for cancer, vascular remodeling, acute inflammation, and rheumatoid arthritis (11,12).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.