Western blot analysis of insulin treated (100 nM, 5 min) MCF7 and C2C12 cells using Phospho-IRS-1 (Ser302) (34C7) Rabbit mAb.
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 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 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 C2C12 cells, untreated or insulin-treated (100 nM, 5 min) using, Phospho-IRS-1 (Ser636/639) Antibody.
Immunprecipitation of Phospho-IRS-1 from insulin treated MCF7 cell extracts using Phospho-IRS-1 (Ser1101) antibody (Lane 1). Lane 2: No antibody control. Lane 3: Input control.
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.
Western blot analysis of IRS-1 (#2382) antibody-immunoprecipitated pellets from differentiated C2C12 cells, unstimulated, insulin-stimulated (100 nM for 15 min) or PMA-stimulated (200 mM for 30 min), using Phospho-IRS-1 (Ser1101) Antibody (upper) or IRS-1 Antibody #2382 (lower).
|Phospho-IRS-1 (Ser302) (34C7) Rabbit mAb 2491||20 µl||
||H M||180||Rabbit IgG|
|Phospho-IRS-1 (Ser307) Antibody 2381||20 µl||
||H M R||180||Rabbit|
|Phospho-IRS-1 (Ser318) (D51C3) Rabbit mAb 5610||20 µl||
||H M||180||Rabbit IgG|
|Phospho-IRS-1 (Ser612) (C15H5) Rabbit mAb 3203||20 µl||
||H M R||180||Rabbit IgG|
|Phospho-IRS-1 (Ser636/639) Antibody 2388||20 µl||
||H M R||180||Rabbit|
|Phospho-IRS-1 (Ser1101) Antibody 2385||20 µl||
||H M R||180||Rabbit|
|IRS-1 (D23G12) Rabbit mAb 3407||20 µl||
||H Mk M R||180||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The IRS-1 Inhibition Antibody Sampler Kit provides an economical means to evaluate insulin signaling negative feedback loops via phosphorylation of various IRS-1 serine residues. The kit includes enough antibody to perform two western blot experiments with each primary antibody.
Each activation state antibody recognizes the phosphorylated form of its target. All target residues are based on the sequence for mouse IRS-1, except Ser636/339 and Ser1101, which are based on the sequence for human IRS-1. IRS-1 (D23G12) Rabbit mAb recognizes total IRS-1 protein independent of its phosphorylation state.
Polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding mouse Ser307 (human Ser312), human Ser636/639 (mouse Ser632/635), and human Ser1101 (mouse Ser1097) of IRS-1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding mouse Ser302 (human Ser307), mouse Ser318 (human Ser323), and mouse Ser612 (human 616) of IRS-1. IRS-1 (D23G12) rabbit monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the sequence surrounding Ser270 of human IRS-1 (mouse 1097).
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, many of which are related to negative feedback loops activated during insulin signaling. Ser302 (human Ser307) of IRS-1 is regulated by FOX01 (5), IKKγ, and MYO1C (6). Ser307 (human Ser312) of IRS-1 is phosphorylated by JNK (7) and IKK (8). PKC phosphorylates mouse IRS-1 at Ser318 (human Ser323) by insulin receptor activation or by other stimulation such as TPA, IL-6, and retinoic acid treatment (9-12). The PKC and mTOR pathways mediate phosphorylation of IRS-1 at Ser612 (human Ser616) and Ser632/635 (human Ser636/639), respectively (13,14). Phosphorylation of IRS-1 at Ser1097 (human 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 (15).
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