Immunofluorescence analysis of Phospho-IRS1 pSer307 was done on 70% confluent log phase T-47D cells. 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 Phospho-IRS1 pSer307 Rabbit Polyclonal Antibody (PA11054) at 1ug/ml in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic phosphopeptide corresponding to residues E(301) S I T A T (pS) P A S M V G G K(315) of human IRS-1.|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||1-2 µg/ml|
|Immunofluorescence (IF)||1-2 µg/ml|
|Western Blot (WB)||1-3 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 2 publications below|
PA1-1054 detects phosphorylated IRS-1 (Ser307) in mouse and human cells.
PA1-1054 has been successfully used in Western Blot procedures. By WB, this antibody detects an ~170 kDa protein representing phospho-IRS-1 (Ser307) from 3T3 L1 cells treated with insulin for 10 minutes.
The PA1-1054 immunogen is a synthetic phosphopeptide corresponding to residues E(301) S I T A T (pS) P A S M V G G K(315) of human IRS-1. This peptide is 100% conserved in rats and mice. This peptide (Cat. #PEP-184) is available for use in neutralization and control experiments.
Insulin receptor substrates (IRS) are responsible for several insulin related activities, such as glucose homeostasis, cell growth, cell transformation, apoptosis and insulin signal transduction. Serine/threonine phosphorylation of IRS-1 has been demonstrated to be a negative regulator of insulin signaling and is responsible for its degradation, although IRS-1 degradation pathways are not well understood. IRS-1 has also been shown to be constitutively activated in cancers such as breast cancer, Wilm and quote;s tumors, and adrenal cortical carcinomas, thus making IRS-1 phosphorylation and subsequent degradation an attractive therapeutic option. To date there have been four subtypes identified: IRS-1,2,3, and 4, with IRS-1 being widely expressed.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Enhanced gastrointestinal expression of cytosolic malic enzyme (ME1) induces intestinal and liver lipogenic gene expression and intestinal cell proliferation in mice.
PA1-1054 was used in western blot to identify the role of intestinal cytosolic malic enzyme 1
|Al-Dwairi A,Brown AR,Pabona JM,Van TH,Hamdan H,Mercado CP,Quick CM,Wight PA,Simmen RC,Simmen FA||PloS one (9:null)||2014|
The hyperglycemia-induced inflammatory response in adipocytes: the role of reactive oxygen species.
PA1-1054 was used in western blot to study the important role of reactive oxygen species in the hyperglycemia-induced inflammatory response in adipocytes.
|Lin Y,Berg AH,Iyengar P,Lam TK,Giacca A,Combs TP,Rajala MW,Du X,Rollman B,Li W,Hawkins M,Barzilai N,Rhodes CJ,Fantus IG,Brownlee M,Scherer PE||The Journal of biological chemistry (280:4617)||2005|