1/200 - 1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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.
Highly expressed in placenta, lung, kidney, testis and ovary. Weakly expressed in spleen and thymus. Not expressed in peripheral blood lymphocytes. Detected in hippocampus.
Rel Similarity Domain (RSD) allows DNA-binding and cooperative interactions with AP1 factors.
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'.
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.
ICC/IF image of ab99431 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab99431 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Immunoprecipitation - NFATC4 antibody (ab99431)
Immunoprecipitation : 1 mg of HeLa cell lysate immunoprecipitated with ab99431 at 3 µg/mg lysate; 20% of immunoprecipitate loaded in lane
Western Blot: NFAT3 antibody (ab99431) at 0.4 µg/ml
developed using the ECL technique
Exposure time : 10 seconds
Predicted band size : 95 kDa
Western blot - NFATC4 antibody (ab99431)
All lanes : Anti-NFATC4 antibody (ab99431) at 0.04 µg/ml
Lane 1 : HeLa whole cell lysate at 50 µg Lane 2 : HeLa whole cell lysate at 15 µg Lane 3 : HeLa whole cell lysate at 5 µg Lane 4 : 293T whole cell lysate at 50 µg Lane 5 : NIH3T3 whole cell lysate at 50 µg