- 44-528G - Invitrogen Antibodies EIF4E antibody

Submitted references Sinoporphyrin sodium is a promising sensitizer for photodynamic and sonodynamic therapy in glioma.

An YW, Liu HQ, Zhou ZQ, Wang JC, Jiang GY, Li ZW, Wang F, Jin HT
Oncology reports 2020 Oct;44(4):1596-1604

Identification of DNA response elements regulating expression of CCAAT/enhancer-binding protein (C/EBP) β and δ and MAP kinase-interacting kinases during early adipogenesis.

Merrett JE, Bo T, Psaltis PJ, Proud CG
Adipocyte 2020 Dec;9(1):427-442

Cotargeting MNK and MEK kinases induces the regression of NF1-mutant cancers.

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 2016 Jun 1;126(6):2181-90

Autocrine Semaphorin3A stimulates eukaryotic initiation factor 4E-dependent RhoA translation in breast tumor cells.

Pan H, Bachelder RE
Experimental cell research 2010 Oct 15;316(17):2825-32

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (20 µg lysate) of HeLa (lane 1), Serum Starved HeLa (lane 2), HeLa Serum Starved for overnight followed by Serum Released (lane 3), HEK-293 (lane 4), HEK-293 treated for 30 minutes with 25 µg/mL of Anisomycin (lane 5), L6 (lane 6) and L6 treated for 10 minutes with 200 ng/mL of Insulin (lane 7). The blots were probed with Anti-eIF4E (pS209) Rabbit Polyclonal Antibody (Product # 44-528G, 1:500-1:2000 dilution) and detected by chemiluminescence Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Two bands ~ 25 and 28 kDa band corresponding to eIF4E (pS209) was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (20 µg lysate) of HeLa (lane 1), Serum Starved HeLa (lane 2), HeLa Serum Starved for overnight followed by Serum Released (lane 3), HEK-293 (lane 4), HEK-293 treated for 30 minutes with 25 µg/mL of Anisomycin (lane 5), L6 (lane 6) and L6 treated for 10 minutes with 200 ng/mL of Insulin (lane 7). The blots were probed with Anti-eIF4E (pS209) Rabbit Polyclonal Antibody (Product # 44-528G, 1:500-1:2000 dilution) and detected by chemiluminescence Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Two bands ~ 25 and 28 kDa band corresponding to eIF4E (pS209) was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Antibody-Peptide Competition. Extracts of HeLa cells were resolved by SDS-PAGE on a 4-20% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature, either left untreated (1-4) or treated with lambda phosphatase (5), and then incubated with the eIF-4E (pS209) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the phosphopeptide immunogen (2), a generic phosphoserine-containing peptide (3), or the phosphopeptide immunogen (4). After washing, the membrane was incubated with goat F (ab’)2 anti-rabbit IgG alkaline phosphatase (Product # ALI4405) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to eIF-4E (pS209) blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, further verifying that the antibody is phospho-specific.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 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 (Product # 44-528G) at 1:250 dilution 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 (Product # S36938). F-actin (Panel c: red) was stained with 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of eIF4E [pS209] was done on U-87 MG cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with eIF4E [pS209] Rabbit Polyclonal Antibody (44528G, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: RNA immunoprecipitation (RIP) western of EIF4E pSer209 was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of EIF4E pSer209 polyclonal antibody (Product # 44-528G) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti- rabbit coated Dynabeads (Product # 11204D), washed extensively, and eluted with NuPAGE™ LDS Sample Buffer (Product # NP0007). Samples were resolved onto NuPAGE™ 4-12% Bis-Tris gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to PVDF membrane (Product # IB23001). Membrane was blocked in 5% milk. Target was detected using a EIF4E pSer209 polyclonal antibody (Product # 44-528G) at a dilution of 1:2000, followed by a 1:4000 dilution of secondary antibody. Chemiluminescent detection was performed using ECL Western Blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. (a,c) 3T3-L1 fibroblasts were treated with differentiation medium for the indicated times (h or min, unless indicated by D = days) at which point cells were harvested and analysed by immunoblot using the indicated antibodies. Data are representative of at least three independent experiments. (b) Day 9 adipocytes were stained with ORO to assess extent of differentiation and lipid accumulation; representative image shown, scale bar 0.2 um

44-528G

Antibody data