Immunofluorescence analysis of Phospho-eIF4E pSer209 was done on 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho eIF4E pSer209 Rabbit Polyclonal Antibody (44528G) at 2ug/ml in 0.1% 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 cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse, Rabbit, Rat|
|Published species reactivity||Human, Not Applicable|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human eIF4E that contains serine 209. The sequence is conserved in mouse, rat and rabbit.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2-3 ug/ml|
|Immunofluorescence (IF)||2-3 ug/ml|
|Western Blot (WB)||Assay Dependent|
* 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|
All eukaryotic cellular mRNAs are blocked at their 5-prime ends with the 7-methylguanosine cap structure, m7GpppX (where X is any nucleotide). This structure is involved in several cellular processes including enhanced translational efficiency, splicing, mRNA stability, and RNA nuclear export. EIF4E is a eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs. It is a 24-kD polypeptide that exists as both a free form and as part of a multiprotein complex termed EIF4F. The EIF4E polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. The other subunits of EIF4F are a 50-kD polypeptide, termed EIF4A (see MIM 601102), that possesses ATPase and RNA helicase activities, and a 220-kD polypeptide, EIF4G. (Rychlik et al., 1987 [PubMed 3469651]).
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Not Applicable||Not Cited||
Cotargeting MNK and MEK kinases induces the regression of NF1-mutant cancers.
44-528G was used in western blot to analyze induction of the regression of NF1-mutant cancers by cotargeting MNK and MEK kinases
|Lock R,Ingraham R,Maertens O,Miller AL,Weledji N,Legius E,Konicek BM,Yan SC,Graff JR,Cichowski K||The Journal of clinical investigation (126:2181)||2016|
|Human||Not Cited||Autocrine Semaphorin3A stimulates eukaryotic initiation factor 4E-dependent RhoA translation in breast tumor cells.||Pan H,Bachelder RE||Experimental cell research (316:2825)||2010|