Western blot analysis of MCF-7 cell extracts, unstimulated and insulin-stimulated (100 nM for 5 min), using IRS-1 Antibody #2382 (left) and Phospho-IRS-1 (Ser307) Antibody (right).
Western blot analysis of cell extracts from CHO IR/IRS-1 cells, untreated or treated with insulin, using Phospho-IRS-1 (Ser612) (C15H5) Rabbit mAb (upper and middle) or IRS-1 Antibody #2382 (lower). The middle blot was treated with calf intestinal phosphatase (CIP) before antibody probing.
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 (top) or IRS-1 Antibody #2382 (bottom).
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 (upper), or IRS-1 Antibody #3407 (lower).
Western blot analysis of MCF7, C2C12 and RD cell lines using IRS-1 (D23G12) Rabbit mAb.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from CHO IR/IRS-1 cells (transfected with insulin receptor and IRS-1), untreated or insulin-treated (100 nM for 5 min), showing an increase in phospho-IRS-1 (Ser307) with insulin stimulation, using Phospho-IRS-1 (Ser307) Antibody.
|Phospho-IRS-1 (Ser307) Antibody 2381||20 µl||
||H M R||180||Rabbit|
|Phospho-IRS-1 (Ser612) (C15H5) Rabbit mAb 3203||20 µl||
||H M R||180||Rabbit IgG|
|IRS-2 Antibody 4502||20 µl||
||H M R||185||Rabbit|
|Phospho-IRS-1 (Ser318) (D51C3) Rabbit mAb 5610||20 µl||
||H M||180||Rabbit IgG|
|IRS-1 (D23G12) Rabbit mAb 3407||20 µl||
||H M R Mk||180||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
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 two western blots with each primary antibody.
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.
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.
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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