Invitrogen

Phospho-IR/IGF1R (Tyr1162, Tyr1163) Polyclonal Antibody

Advanced Verification

This Antibody was verified by Cell treatment to ensure that the antibody binds to the antigen stated. View Details

9 Published Figures

21 References

View all (8) IR/IGF1R antibodies

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Datasheet

Protocols

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Phospho-IR/IGF1R (Tyr1162, Tyr1163) Antibody in Western Blot (WB) — Upregulation and Antibody-Peptide Competition. Extracts of CHO-T cells over-expressing the human insulin receptor unstimulated (1) or stimulated with 100 nM insulin for 10 min at 37°C (2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C and incubated with the IR/IGF1R (pYpY1162/1163) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the phosphopeptide immunogen (2), the non-phosphorylated peptide corresponding to the phosphopeptide immunogen (3), or a generic phosphotyrosine-containing peptide (4). After washing, the membrane was incubated with goat F (ab')2 anti-rabbit IgG HRP-conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™method. The data show that only the phosphopeptide corresponding to IR/IGF1R (pYpY1162/1163) completely blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the up-regulation of this site upon stimulation with insulin in this cell system.

Phospho-IR/IGF1R (Tyr1162, Tyr1163) Antibody in Immunocytochemistry (ICC/IF) — Immunofluorescence analysis of IR/IGF1R (pYpY1162/1163) was done on 70% confluent log phase MCF7 cells treated with insulin (100nM for 5 min). The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with IR/IGF1R (pYpY1162/1163) Rabbit polyclonal Antibody (Product # 44-804G) at 2 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing membrane localization. Panel e shows untreated MCF7 cells. Panel f shows no primary antibody control. The images were captured at 20X magnification.

Phospho-IR/IGF1R (Tyr1162, Tyr1163) Antibody in Cell treatment — Modulation of target protein phosphorylation by cell treatment demonstrates antibody specificity. Immunofluorescence analysis of IR/IGF1R pY1162pY1163 using IR/IGF1R (pY1162pY1163) polyclonal antibody (Product # 44-804G) shows induced expression of IR/IGF1R (pY1162pY1163) in the membrane of MCF7 cells treated with insulin. {TM}

Phospho-IR/IGF1R (Tyr1162, Tyr1163) Antibody — Compound 23 increases liver cell insulin signaling, is orally bioavailable and reverses diabetes in obese mice. ( a , b ) HepG2 hepatocytes were incubated overnight with 10 muM compound or DMSO and stimulated with 10 nM insulin for 5 min or left unstimulated (unst.). Insulin receptor (IR) tyrosine phosphorylation was assessed by ( a ) western blotting (WB) of anti-IR immunoprecipitations (representative of two independent experiments) and ( b ) phosphoIR (pIR) ELISA (data from five independent experiments. 23 , P = 0.0079; 28 , P = 0.1667). ( c - g ) Diet-induced obese (DIO) male B6 mice were treated with 0.05% (w/w) 23 in high-fat diet (HFD) or HFD alone for 2 weeks. ( c ) Body weight during treatment (HFD, n = 17; 23 , n = 13). ( d ) Intraperitoneal glucose tolerance test (IPGTT); P = 0.0162 (HFD, n = 8; 23 , n = 6). ( e ) Fasting plasma insulin levels, relative to littermates fed HFD; P = 0.0235 (HFD, n = 4; 23 , n = 4). ( f - g ) Mice were injected intraperitoneally with insulin and livers harvested after 10 min. ( f ) Liver IR tyrosine phosphorylation; P = 0.0002 (HFD, n = 7; 23 , n = 6). ( g ) Western blot of liver homogenates (HFD, n = 3; 23 , n = 3). ( h , i ) DIO liver-specific LMPTP KO mice were treated with 23 (HFD, n = 6; 23 , n = 6) as in c-g . ( h ) IPGTT, P = 0.6120. ( i ) Liver IR tyrosine phosphorylation; P = 0.2791. For b - f , h , i mean +- s.e.m. is shown; *, P < 0.05; NS, nonsignificant ( P >= 0.05); b , Kolmogorov-Smirnov test; d , h , two-way ANOV {TM}

Product Details

Applications

Tested Dilution

Publications

Western Blot (WB)

1:1000

View 18 publications 18 publications

Immunohistochemistry (IHC)

Assay Dependent

View 2 publications 2 publications

Immunohistochemistry (Paraffin) (IHC (P))

View 1 publication 1 publication

Immunocytochemistry (ICC/IF)

1:100-1:500

Product Specifications

Species Reactivity

Human

Published species

Human, Mouse, Rat

Host/Isotype

Rabbit / IgG

Class

Polyclonal

Type

Antibody

Immunogen

Synthetic phosphopeptide derived from the region of IR/IGF1R that contains tyrosines 1162 and 1163 of the human insulin receptor (IR)

Conjugate

Unconjugated

Form

Liquid

Purification

Antigen affinity chromatography

Storage buffer

Dulbecco's PBS, pH 7.3, with 1mg/mL BSA, 50% glycerol

Contains

0.05% sodium azide

Storage conditions

-20°C

RRID

AB_2533762

Product Specific Information

The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody reactive with non-phosphorylated insulin/insulin-like growth factor-1 receptor (IR/IGF1R). The final product is generated by affinity chromatography using an IR/IGF1R-derived peptide that is phosphorylated at tyrosines 1162 and 1163 (tyrosines 1135 and 1136 for IGF1R. The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of IR/IGF1R that contains tyrosines 1162 and 1163 of the human insulin receptor (IR) as numbered according to Ebina, et al. (1150 and 1151 according to Ullrich et al.). The corresponding residues in the IGF1R are 1135 and 1136. The sequence is conserved in mouse and rat for both the IR and IGF1R. Although exhibiting a preference for IR/IGF1R, this antibody has been shown by both peptide competition and protein blotting to react with other dual-phosphotyrosine motifs from proteins such as c-Met and Shc. \r
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This antibody has been used in western blotting and previous lots of this antibody have been used in immunostaining. Positive controls used with this antibody in western analysis were Chinese hamster ovary cells transfected with a vector containing human insulin receptor (CHO-T) and stimulated with insulin, or 3T3-L1 cells stimulated with insulin.

Target Information

Insulin and Insulin-like Growth Factor receptors are transmembrane tyrosine kinase receptors that play critical roles in development, cell growth and metabolism. Insulin/Insulin-like growth factor-1 binding to the extracellular domain of IR and IGFR leads to autophosphorylation of the receptor and activation of the intrinsic tyrosine kinase activity, which allows appropriate substrates to be phosphorylated. Phosphorylation sites that are unique to each receptor presumably play a key role in these signaling differences. The catalytic loops within the tyrosine kinase domains contain a three tyrosine motif corresponding to Tyr1158, 1162 and 1163 (for the IR) and Tyr1131, 1135 and 1136 (for the IGF1R). Autophosphorylation of the tyrosine sites within the activation loop proceeds in a processive manner initiating at the second tyrosine (1162 or 1135), followed by phosphorylation at tyrosines 1158 (IGF-1R tyrosine 1131), 1163 (IGF-1R tyrosine 1136), resulting in the full activation of IR and IGF1R.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: CD220; CD221; IGF-I receptor; IGF-I Receptor beta; IGF1 Receptor; IGF1 Receptor beta; IGF1R beta; Insulin receptor; Insulin receptor subunit alpha; Insulin receptor subunit beta; Insulin-like growth factor 1 receptor; Insulin-like growth factor 1 receptor alpha chain; Insulin-like growth factor 1 receptor beta chain; Insulin-like growth factor I receptor; IR; soluble IGF1R variant 1; soluble IGF1R variant 2

Gene Aliases: CD220; CD221; HHF5; IGF1R; IGFIR; IGFR; INSR; JTK13

UniProt ID: (Human) P08069, (Human) P06213

Entrez Gene ID: (Human) 3480, (Human) 3643

Function(s)