Figure 1. Treatment of hSkMC cells with insulin stimulates phosphorylation of IRS-1 at Ser302, detected by the PathScan® Phospho-IRS-1 (Ser302) Sandwich ELISA Kit #7283, but does not affect the level of total IRS-1 detected by PathScan® Total IRS-1 Sandwich ELISA Kit #7328. hSkMC cells (80-90% confluent) were starved overnight and treated with 100 nM insulin for 15 minutes at 37oC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots using IRS-1 (L3D12) Mouse mAb #3194 (left panel) or Phospho-IRS-1 (Ser302) (34C7) Rabbit mAb #2491 (right panel) are shown in the bottom figure.
Figure 2. The relationship between the protein concentration of the lysate from untreated and insulin-treated hSkMC cells and the absorbance at 450 nm is shown.
The PathScan® Phospho-IRS-1 (Ser302) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IRS-1 when phosphorylated at Ser302. An IRS-1 Mouse Antibody* has been coated onto the microwells. After incubation with cell lysates, IRS-1 (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Phospho-IRS-1 (Ser302) Rabbit Detection Antibody* is added to detect phosphorylation of Ser302 on the captured IRS-1 protein. Anti-rabbit IgG, HRP-linked Antibody #7074* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of IRS-1 phosphorylated at Ser302.
* Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Phospho-IRS-1 (Ser302) Sandwich ELISA Kit #7283 detects endogenous levels of IRS-1 when phosphorylated at Ser302. As shown in Figure 1, a significant induction of IRS-1 phosphorylation at Ser302 can be detected in hSkMC cells following treatment with insulin using the Phospho-IRS-1 (Ser302) Sandwich ELISA Kit #7283. The level of total IRS-1 (phospho and nonphospho) remains unchanged as shown by Western analysis and by PathScan® Total IRS-1 Sandwich ELISA Kit #7328 (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Insulin receptor substrate 1 (IRS-1) is one of the major substrates of the insulin receptor kinase (1). IRS-1 contains multiple tyrosine phosphorylation motifs that serve as docking sites for SH2-domain containing proteins that mediate the metabolic and growth-promoting functions of insulin (2-4). IRS-1 also contains over 30 potential serine/threonine phosphorylation sites. Ser307 of IRS-1 is phosphorylated by JNK (5) and IKK (6) while Ser789 is phosphorylated by SIK-2, a member of the AMPK family (7). The PKC and mTOR pathways mediate phosphorylation of IRS-1 at Ser612 and Ser636/639, respectively (8,9). Phosphorylation of IRS-1 at Ser1101 is mediated by PKCθ and results in an inhibition of insulin signaling in the cell, suggesting a potential mechanism for insulin resistance in some models of obesity (10).
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