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Immunofluorescence analysis of 4E-BP1 was done on 70% confluent log phase HeLa 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 4E-BP1 Mouse Monoclonal Antibody (AHO1382) at 2 µg/mL and incubated for 3 hours at room temperature and then labeled with Alexa Flour 488 Rabbit Anti-Mouse IgG Secondary Antibody (A11059) 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 with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (A12381). Panel d is a merged image showing nuclear and perinuclear localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.
|Tested species reactivity||Human , Rat , Mouse|
|Host / Isotype||Mouse / IgG1, kappa|
|Immunogen||Recombinant human 4E-BP1 protein expressed in E. coli.|
|Storage buffer||PBS, pH 7.2, with 1% BSA|
|Contains||0.1% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:100-1:500|
|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.
Eukaryotic initiation factor 4E binding protein 1 (4E-BP1), also known as PHAS, is a ~20 kDa member of a family of eIF4E-binding proteins whose binding affinity to eIF4E is regulated by its phosphorylation. It inhibits cap-dependent translation by binding to eIF4E on the same site that overlaps the binding site for eIF4G, preventing its binding to the latter and eventually leading to an increase in mRNA translation. The phosphorylation of 4E-BP1 is critical in determining cell fate by controlling translation initiation and apoptotic potency. 4E-BP1 is hyperphosphorylated in response to several external stimuli including hormones, growth factors, mitogens, cytokines and G-protein–coupled receptors and in response to stress conditions including nutrient deprivation. The phosphorylation of these sites is believed to occur in an orderly fashion where phosphorylation of threonine 37 and 46 by FRAP/mTOR is a priming step for subsequent phosphorylation of 4E-BP1 at the carboxy-terminal sites. Under normoxic conditions, increased VEGF expression, resulting from inhibition of 4E-BP1, contributes to efficient angiogenesis and metastatic brain growth through activated integrin alphav beta3.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
AA959816, PHAS-I, 4EBP1, 4e-bp1, 4E-BP1, BP-1
eIF4E-binding protein 1, eukaryotic translation initiation factor 4E-binding protein 1, phosphorylated heat- and acid-stable protein regulated by insulin 1, 4E-BP1, 4EBP1, EIF4EBP1, Eukaryotic translation initiation factor 4E binding protein 1, Insulin-stimulated EIF-4E binding protein PHAS-I, P/OKCL.6, PHAS-1, PHAS-I