Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab3447 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000. Detects a band of approximately 95 kDa (predicted molecular weight: 99 kDa).Can be blocked with NFATC4 peptide (ab4979).
IHC-FoFr Use at an assay dependent concentration. PubMed: 20530871
ICC/IF 1/100.
IHC-P 1/3000.
IP Use at an assay dependent concentration.

3 μg

Target

  • Function
    Plays a role in the inducible expression of cytokine genes in T-cells, especially in the induction of the IL-2 and IL-4. Transcriptionally repressed by estrogen receptors; this inhibition is further enhanced by estrogen. Increases the transcriptional activity of PPARG and has a direct role in adipocyte differentiation. May play an important role in myotube differentiation. May play a critical role in cardiac development and hypertrophy. May play a role in deafferentation-induced apoptosis of sensory neurons.
  • Tissue specificity
    Highly expressed in placenta, lung, kidney, testis and ovary. Weakly expressed in spleen and thymus. Not expressed in peripheral blood lymphocytes. Detected in hippocampus.
  • Sequence similarities
    Contains 1 IPT/TIG domain.
    Contains 1 RHD (Rel-like) domain.
  • Domain
    Rel Similarity Domain (RSD) allows DNA-binding and cooperative interactions with AP1 factors.
  • Post-translational
    modifications
    Phosphorylated by NFATC-kinases; dephosphorylated by calcineurin. Phosphorylated on Ser-168 and Ser-170 by MTOR, IRAK1, MAPK7 and MAPK14, on Ser-213 and Ser-217 by MAPK8 and MAPK9, and on Ser-289 and Ser-344 by RPS6KA3. Phosphorylated by GSK3B.
    Ubiquitinated, leading to its degradation by the proteasome and reduced transcriptional activity. Ubiquitination and reduction in transcriptional activity can be further facilitated through GSK3B-dependent phosphorylation. Polyubiquitin linkage is mainly through 'Lys-48'.
  • Cellular localization
    Cytoplasm. Nucleus. Cytoplasmic for the phosphorylated form and nuclear after activation that is controlled by calcineurin-mediated dephosphorylation. Rapid nuclear exit of NFATC is thought to be one mechanism by which cells distinguish between sustained and transient calcium signals. The subcellular localization of NFATC plays a key role in the regulation of gene transcription.
  • Information by UniProt
  • Database links
  • Alternative names
    • cytoplasmic 4 antibody
    • NF ATc4 antibody
    • NF-AT3 antibody
    • NF-ATc4 antibody
    • NFAC4_HUMAN antibody
    • NFAT3 antibody
    • NFATc4 antibody
    • Nuclear factor of activated T cells cytoplasmic 4 antibody
    • Nuclear factor of activated T cells cytoplasmic calcineurin dependent 4 antibody
    • Nuclear factor of activated T-cells antibody
    • T cell transcription factor NFAT3 antibody
    • T-cell transcription factor NFAT3 antibody
    see all

Images

  • Immunocytochemistry/Immunofluorescence analysis of NFATC4 in A431 cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab3447 at a dilution of 1:100 overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. NFATC4 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of NFATC4 in HeLa cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab3447 at a dilution of 1:20 overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. NFATC4 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of NFATC4 in U251 cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control - right) or with ab3447 at a dilution of 1:20 overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. NFATC4 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

  • All lanes : Anti-NFATC4 antibody (ab3447) at 1/1000 dilution

    Lane 1 : MCF7 whole cell lysate
    Lane 2 : Jurkat whole cell lysate
    Lane 3 : Raji whole cell lysate
    Lane 4 : Ramos whole cell lysate
    Lane 5 : HepG2 whole cell lysate
    Lane 6 : U2OS whole cell lysate
    Lane 7 : HeLa whole cell lysate

    Lysates/proteins at 25 µg per lane.

    Secondary
    All lanes : HRP conjugated Goat anti-rabbit at 1/20000 dilution

    Predicted band size: 99 kDa

  • Immunoprecipitation of NFATC4 was performed on MCF7 cells. The antigen:antibody complex was formed by incubating 500µg whole cell lysate with 3µg of rabbit polyclonal antibody recognizing NFATC4 (ab3447) overnight on a rocking platform at 4°C. The immune-complex was captured on 50µl Protein A/G Agarose. Captured immune-complexes were washed and proteins eluted with 5X Reducing Sample Loading Dye . Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to PVDF membrane and blocked with 5% Milk/TBS-0.1%Tween for at least 1 hour. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody at a dilution of 1:20000 for at least one hour. Membranes were washed and chemiluminescent detection performed.



    All lanes :

    Lane 1 : MCF7 cell lysate
    Lane 2 : Immunoprecipitate

    Secondary
    All lanes : HRP-conjugated Goat anti-rabbit at 1/20000 dilution
  • Immunofluorescent analysis of NFATC4 using anti-NFATC4 polyclonal antibody ( ab3447) (shown in green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were then blocked with 1% BSA for 15 minutes at room temperature. Cells were probed with a rabbit polyclonal antibody recognizing NFATC4 ( ab3447) at a dilution of 1:100 for at least 1 hour at room temperature. Cells were washed with PBS and incubated with DyLight 488 goat-anti-rabbit secondary antibody at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

References

This product has been referenced in:
  • Dittmann K  et al. New roles for nuclear EGFR in regulating the stability and translation of mRNAs associated with VEGF signaling. PLoS One 12:e0189087 (2017). Read more (PubMed: 29253018) »
  • Balakrishnan A  et al. Temporal Analysis of Gene Expression in the Murine Schwann Cell Lineage and the Acutely Injured Postnatal Nerve. PLoS One 11:e0153256 (2016). IHC . Read more (PubMed: 27058953) »
See all 3 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

BATCH NUMBER 306063 ORDER NUMBER 230325 DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE human B cell lymphoma cell line whole cell extract PRIMARY ANTIBODY Abcam anti-NFATc4 was used Recommended dilution 1:10 000, as well as 1:5000 used Antibody was diluted in TBST with 1% BSA, and membrane was incubated at 4 deg celcius overnight Membrane was then washed with TBST thrice, 10 min each time on shaker DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Bands obtained using same sample with Santa Cruz's anti-NFATc4 ANTIBODY STORAGE CONDITIONS Antibody was aliquoted and stored at -30 deg celcius upon arrival SAMPLE PREPARATION Proteins were extracted using RIPA buffer with protease inhibitors. Protein was heated with 4x loading dye at 95 deg celcius for 5 mins, before being loaded. AMOUNT OF PROTEIN LOADED 40ug ELECTROPHORESIS/GEL CONDITIONS 6% SDS-PAGE gel used. 80V was used initially when proteins were migrating through the stacking gel, and increased to 140V thereafter. TRANSFER AND BLOCKING CONDITIONS Transfer to PVDF memebrane was carried out at 4 deg celcius overnight at 30V. Transfer buffer composition [2.9g glycine, 5.8g Tris, 0.37g SDS, pH8.3] Membrane was blocked in 5% BSA for 1 h at room temperature. SECONDARY ANTIBODY Santa Cruz Biotechnology goat-anti-rabbit IgG-HRP used Dilution 1:1000 in TBST with 1% BSA Membrane was incubated for 1 h at room temperature, and subsequently washed thrice in TBST, 10 min per wash with agitation HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? As recommended in answer for previous enquiry CCE1098662 - using a human protein sample and replacing the milk with BSA

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Answer

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab3447 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. I have looked through the protocols you used and would like to confirm the following items. - can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working. - could the no signal you are experiencing be due to poor transfer of protein to membrane? Did you check the transfer efficiency? This can be simply done with a reversible stain such as Ponceau S. I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, I will be more than happy to offer you a replacement or refund. In which case, please confirm your shipping address/purchasing agent information. Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund for you.

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Answer

Thank you for your enquiry. Unfortunately I do not have details of specific antigen retrieval that should be performed for this antibody. However, may I recommend you start with the mildest form of antigen retrieval in the form of heat mediated antigen retrieval. A time course should be performed to determine the best conditions to present the antigen to the antibody. We have excellent protocols located at the following web link: https://www.abcam.com/index.html?pageconfig=antigenretrieval I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Question
Answer

According to the datasheet, this antibody has not yet been tested in mouse. We will update the on-line datasheet of this product as soon as get more data.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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