710880

antibody from Invitrogen Antibodies

Targeting: FOXO4 AFX1, MLLT7

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

710880

Provider

Invitrogen Antibodies

Product name

Phospho-FOXO4 (Thr28) Recombinant Polyclonal Antibody (12HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Goat, Pig, Bovine, Dog, Rat and Mouse.

Antibody clone number

12HCLC

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of A431 (Lane 1), A431 treated with PDGF (150 nM/10 min) (Lane 2), A431 treated with EGF (200 ng/mL for 10 min) (Lane 3). The blots were probed with Anti-FoxO4 (pT28) Recombinant Rabbit Polyclonal Antibody (Product # 710880, 1-3 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A clear 55 kDa band corresponding to FoxO4 (pT28) was observed across cell lines tested.B) To confirm the specificity of Anti-FoxO4 (pT28) Recombinant Rabbit Polyclonal Antibody, competition was performed with the phosphopeptide (10 µg/mL) as shown in the corresponding blot on right. The peptide competes with the antibody and prevents it from binding to the membrane. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane by overnight wet transfer method. 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).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence was performed on fixed and permeabilized A431 cells for detection of FoxO4 (pT28) using Anti-FoxO4 (pT28) Recombinant Rabbit Polyclonal Antibody (Product # 710880, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Panel a) shows representative cells that were stained for detection and localization of FoxO4 (pT28) (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic and nuclear localization of FoxO4 (pT28). Panel e) represents control cells with no primary antibody to assess background.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry analysis of Foxo4 showing staining in the nucleus of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Foxo4 Recombinant Rabbit Polyclonal Antibody (Product # 710880) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.