710323

antibody from Invitrogen Antibodies

Targeting: EIF4EBP1 4E-BP1, PHAS-I

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

710323

Provider

Invitrogen Antibodies

Product name

Phospho-4EBP1 (Thr46) Recombinant Polyclonal Antibody (19HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with mouse, rat, non-human primate and rabbit based on sequence homology.

Antibody clone number

19HCLC

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of 4EBP1 (pT46) was performed by loading 30 µg of HEK-293 (lane1), HEK-293 treated for 10 minutes with 100 nM/mL of EGF (lane2), MCF7 (lane3), and MCF7 treated for 15 minutes with 150 µg/mL of Insulin (lane4) 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. 4EBP1 (pT46) was detected at ~20 kDa using 4EBP1 (pT46) Recombinant Rabbit Polyclonal Antibody (Product # 710323) at 1-2 µg/mL in 2.5% skim milk at 4°C overnight on a rocking platform. 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 of Phospho-4EBP1 pThr46 in whole cells extracts of 293 cells treated with EGF (lane 1) and NIH-3T3 cells treated with PDGF (lane 3) using a Phospho-4EBP1 pThr46 Recombinant Rabbit Polyclonal Antibody (Product # 710323) at a dilution of 2 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lanes 2 and 4). Samples were detected using chemiluminescence (ECL). Results show a band at ~20kDa.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of Phospho-4EBP1 pThr46 in HeLa cells using a Phospho-4EBP1 pThr46 Recombinant Rabbit Polyclonal Antibody (Product # 710323) followed by detection using an Alexa Fluor 488-conjugated Goat anti-Rabbit secondary antibody (green) (Image A). Nuclei were stained using DAPI (Image B) and actin stained with Alexa Fluor 594 phalloidin (red) (image C). Image D is a composite image showing nuclear localization of phosphorylated 4EBP1 and Image E is a composite image of cells showing inhibition of antibody binding after competition with the phosphorylated peptide.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of 4EBP1 (pT46) 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 followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with 4EBP1 (pT46) Recombinant Rabbit Polyclonal Antibody (Product # 710323) at a dilution of 2 µg/mL 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 4EBP1 (pT46). Panel e shows competition with 4EBP1 (pT46) peptide. The images were captured at 20X magnification.