- 700238 - Invitrogen Antibodies EIF4EBP1 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

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), HEK-293 treated with EGF (200 ng/mL for 10 min) (Lane 2) and HEK-293 treated with Rapamycin (20 nM for 30 min) followed by EGF (200 ng/mL for 10 min) (Lane 3). The blot was probed with Anti-Phospho-4EBP1 (Thr37) Recombinant Rabbit Monoclonal Antibody (Product # 700238, 1µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 17 kDa band corresponding to Phospho-4EBP1 (Thr37) was detected and showed increase in phosphorylation on EGF treatment and decrease upon rapamycin treatment.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of 4E-BP1 (pT37) was performed by loading 20 µg of U2OS (lane1) and HEK-293 (lane2) cell lysates using Novex®NuPAGE®4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. 4E-BP1 (pT37) was detected at ~20 kDa using 4E-BP1 (pT37) Recombinant Rabbit Monoclonal Antibody (Product # 700238) at 1 µg-3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with phosphopeptide (10 µg/mL) as shown in the corresponding blot on right. Goat anti-Rabbit IgG-HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and 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 (30 µg lysate) of HEK-293 (Lane 1), HEK-293 treated with EGF (200 ng/mL for 10 min) (Lane 2) and HEK-293 treated with Rapamycin (20 nM for 30 min) followed by EGF (200 ng/mL for 10 min) (Lane 3). The blot was probed with Anti-Phospho-4EBP1 (Thr37) Recombinant Rabbit Monoclonal Antibody (Product # 700238, 1µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4,000 dilution). A 17 kDa band corresponding to Phospho-4EBP1 (Thr37) was detected and showed increase in phosphorylation on EGF treatment and decrease upon rapamycin treatment.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phospho-4EBP1 pThr37 in HeLa cells using a Phospho-4EBP1 pThr37 recombinant rabbit monoclonal antibody (Product # 700238) at a dilution of 2.5 µg/mL in the absence of peptide (top left) and presence of phosphopeptide used as immunogen (top right) or non-phosphopeptide (bottom left), followed by detection using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at a dilution of 1:1000. Actin was stained with Alexa Fluor 568 phalloidin (Product # A12380).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Phospho-4EBP1 pThr37 in HeLa cells using a Phospho-4EBP1 pThr37 recombinant rabbit monoclonal antibody (Product # 700238) at a dilution of 2.5 µg/mL in the absence of peptide (top left) and presence of phosphopeptide used as immunogen (top right) or non-phosphopeptide (bottom left), followed by detection using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at a dilution of 1:1000. Actin was stained with Alexa Fluor 568 phalloidin (Product # A12380).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of 4E-BP1 (pT37) was done on 70% confluent log phase U2OS cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with 4E-BP1 (pT37) Recombinant Rabbit Monoclonal Antibody (Product # 700238) at 2 µg-4 µg 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 with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (Product # A12381). Panel d is a merged image showing nuclear localization of 4E-BP1 (pT37). Panel e shows competition with 4E-BP1 (pT37) peptide. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Phospho-4EBP1 pThr37 in formalin-fixed, paraffin-embedded human endometrial carcinoma (top left) breast carcinoma (top right) and Hodgkin lymphoma (bottom) using a Phospho-4EBP1 pThr37 monoclonal antibody (Product # 700238) at a dilution of 5 µg/mL. Images were taken at a magnification of 20x (top) or 40x (bottom). Results show strong nuclear staining in tumor cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Phospho-4EBP1 pThr37 in formalin-fixed, paraffin-embedded human endometrial carcinoma (top left) breast carcinoma (top right) and Hodgkin lymphoma (bottom) using a Phospho-4EBP1 pThr37 monoclonal antibody (Product # 700238) at a dilution of 5 µg/mL. Images were taken at a magnification of 20x (top) or 40x (bottom). Results show strong nuclear staining in tumor cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of 4E-BP1 [pT37] was done on HeLa cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with ABfinityª 4E-BP1 [pT37] Recombinant Rabbit Monoclonal Antibody (700238, red histogram) or with rabbit isotype control (pink histogram) at 2 µg-4 µg/million cells in 2.5% BSA. After incubation at room temperature for 2-3 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: Flow cytometry analysis of Phospho-4EBP1 pThr37 in Jurkat cells using a Phospho-4EBP1 pThr37 recombinant rabbit monoclonal antibody (Product # 700238) at a dilution of 0.1 µg. Cells were fixed and permeabilized using FIX & PERM (Product # GAS-004) reagent, and detection was performed using an Alexa Fluor 488 goat anti-rabbit IgG (black) compared to a control without primary antibody (gray). Pre-incubation with the phosphopeptide decreased the signal (blue) whereas incubation with the non-phosphopeptide did not (red).

700238

Antibody data