Cell Signaling Technology

Product Pathways - Metabolism

Insulin Receptor Substrate Antibody Sampler Kit #3015

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-IRS-1 (Ser307) Antibody #2381 40 µl W IP H M R 180 Rabbit
Phospho-IRS-1 (Ser612) (C15H5) Rabbit mAb #3203 40 µl W H M R 180 Rabbit IgG
IRS-2 Antibody #4502 40 µl W H M R 185 Rabbit
Phospho-IRS-1 (Ser318) (D51C3) Rabbit mAb #5610 40 µl W IP H M (R) 180 Rabbit IgG
IRS-1 (D23G12) Rabbit mAb #3407 40 µl W IP H M R Mk 180 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Phospho-IRS-1 (Ser307) Antibody, Phospho-IRS-1 (Ser612) (C15H5) Rabbit mAb and Phospho-IRS-1 (Ser318) Antibody detect endogenous levels of IRS-1 only when phosphorylated at Ser307, Ser612 or Ser318, respectively. Note: Ser318 is the mouse residue; the corresponding human residue is Ser323. IRS-1 Antibody and IRS-2 Antibody detect endogenous levels of total IRS-1 or IRS-2, respectively. The Phospho-IRS-1 (Ser318) antibody cross reacts with a inducible nonspecific band at around 57 kDa.

Western Blotting

Western Blotting

Western blot analysis of extracts from MCF-7 cells, unstimulated or insulin-stimulated (100 nM for 5 min), using IRS-1 Antibody #2382 (left) and Phospho-IRS-1 (Ser307) Antibody #2381 (right).

Western Blotting

Western Blotting

Western blot analysis of MCF7, C2C12 and RD cell lines using IRS-1 (D23G12) Rabbit mAb #3407.

Western Blotting

Western Blotting

Western blot analysis of extracts from CHO IR/4PS cells (transfected with insulin receptor and IRS-2) or CHO IR/IRS-1 cells (transfected with insulin receptor and IRS-1), showing no change in total protein levels with insulin stimulation (100 nM for 5 min), using IRS-2 Antibody #4502 (upper) or IRS-1 Antibody #2382 (lower).


Western Blotting

Western Blotting

Western blot analysis of extracts from serum-starved C2C12 cells, untreated or insulin-treated (150 nM for 5 min.), using Phospho-IRS-1 (Ser318) (D51C3) Rabbit mAb #5610 (upper), or IRS-1 Antibody #3407 (lower).

Description

The Insulin Receptor Substrate Antibody Sampler Kit provides an economical means to investigate IRS-1 and IRS-2 signaling and phosphorylation within the cell. The kit contains enough antibody to perform four western blots with each primary antibody.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy-terminal sequence of human IRS-2 and residues surrounding Ser307 of mouse IRS-1 (homologous to Ser312 of human IRS-1). Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser612 of mouse IRS-1, a synthetic peptide corresponding to the sequence surrounding Ser270 of human IRS-1 and a synthetic phosphopeptide corresponding to residues surrounding Ser318 of mouse IRS-1 protein.

Background

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).

  1. Sun, X.J. et al. (1991) Nature 352, 73-77.
  2. Sun, X.J. et al. (1992) J. Biol. Chem. 267, 22662-22672.
  3. Myers Jr., M.G. et al. (1993) Endocrinology 132, 1421-1430.
  4. Wang, L.M. et al. (1993) Science 261, 1591-1594.
  5. Rui, L. et al. (1997) J. Clin. Invest. 107, 181-189.
  6. Gao, Z. et al. (2002) J. Biol. Chem. 277, 48115-48121.
  7. Horike, N. et al. (2003) J. Biol. Chem. 278, 18440-18447.
  8. Ozes, O.N. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 4640-4645.
  9. De Fea, K. and Ruth, R.A. (1997) Biochemistry 36, 12939-12947.
  10. Li, Y. et al. (2004) J. Biol. Chem. 279, 45304-45307.

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