Antibody Panel to Proteins of the 14-3-3 family (14-3-3 beta, 14-3-3 gamma, 14-3-3 epsilon, 14-3-3 zeta, 14-3-3 theta, 14-3-3 sigma, 14-3-3 eta)

Scientific Background

Protein 14-3-3 is a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases and an endogenous inhibitor of protein kinase C. The 14-3-3 protein exists in several distinct forms: e.g., beta (YWHAB), gamma (YWHAG), epsilon (YWHAE), zeta (YWHAZ), theta (YWHAQ), sigma (SFN, Stratifin), and eta (YWHAH). The highly conserved 14-3-3 proteins are found in both plants and mammals and mediates signal transduction by binding to phosphoserine-containing proteins.

14-3-3-beta interacts with the TSC1-TSC2 dimer. The interaction required phosphorylation of TSC2 at ser1210. Binding of 14-3-3-beta to TSC2 did not alter the interaction between TSC1 and TSC2, but it reduced the ability of the complex to inhibit phosphorylation of ribosomal protein S6 kinase, impairing the ability of the complex to inhibit cell growth.

Cell division cycle (CDC) proteins and cyclin-dependent kinases (CDK) regulate the progression of cells through mitosis, and their activities are regulated by phosphorylation and dephosphorylation. Interacting proteins can also affect CDC and CDK activity. Using a yeast 2-hybrid screen to probe a HeLa cell library with CDC25A and CDC25B as bait, Conklin et al. (1995) isolated cDNAs encoding YWHAB, which they called 14-3-3-beta, and YWHAE, which they called 14-3-3-epsilon. YWHAE encodes a deduced 260-amino acid protein that is 100% identical to the mouse sequence. Both 14-3-3 proteins interacted with either CDC protein but did not affect their phosphatase activities. Like YWHAB, YWHAE interacted with RAF1 but not RAS in yeast 2-hybrid screens and may facilitate the association of CDC25 with RAF1.

The binding of insulin to its receptor induces the phosphorylation of the cytosolic substrates IRS1 and IRS2, which associate with several Src homology-2 (SH2) domain-containing proteins. To identify unique IRS1-binding proteins, Ogihara et al. (1997) screened a human heart cDNA expression library with recombinant IRS1. They obtained 2 isoforms of the 14-3-3 protein family, 14-3-3-zeta (YWHAZ) and -epsilon. 14-3-3 protein has been shown to associate with IRS1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS2, a protein structurally similar to IRS1, was also shown to form a complex with 14-3-3 protein using a baculovirus expression system. The amount of 14-3-3 protein associated with IRS1 was not affected by insulin stimulation but was increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. The authors identified a putative 14-3-3 protein-binding site within the phosphotyrosine-binding (PTB) domain of IRS1. Ogihara et al. (1997) suggested that the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS1.

14-3-3 sigma, called stratifin,is diffusely distributed in the cytoplasm and is present in cultured epithelial cells. It is most abundant in tissues enriched in stratified keratinizing epithelium. 14-3-3-sigma, or stratifin, is strongly induced by gamma irradiation and other DNA-damaging agents. The induction of 14-3-3-sigma is mediated by a p53-responsive element located 1.8 kb upstream of its transcription start site. Exogenous introduction of 14-3-3-sigma into cycling cells results in a G2 arrest.

Rat 14-3-3-gamma (YWHAG) is upregulated in injured rat carotid arteries and 14-3-3 gamma mRNA is upregulated in cytokine-stimulated human vascular smooth muscle cells (VSMC). The deduced 246-amino acid protein, which shares 98% sequence identity with the rat sequence, has preserved 14-3-3 family signature motifs, a predicted annexin motif, and several potential phosphorylation sites but not the CDK2 phosphorylation motif. YWHAG expression is relatively strong in brain, skeletal muscle, and heart but weak in peripheral blood leukocytes. By SDS-PAGE and autoradiographic analysis, Autieri and Carbone (1999) found that 14-3-3 gamma is expressed and phosphorylated by activation with platelet-derived growth factor and other activators of several isoforms of protein kinase C. Inhibitors of PKC block YWHAG phosphorylation. Western blot analysis showed that YWHAG interacts with PKC and with RAF1.

Antibody Panel to proteins of the 14-3-3 family

Acris Antibodies offers a range of antibodies to the 14-3-3 proteins, some of them pan specific, but family member specifc antibodies are available also. Monoclonal and polyclonal antibodies can be used for most immunological techniques like immunohistochemistry, immunoprecipitation and western blotting (see staining pictures below).

Fig.1: 14-3-3 gamma detected in lysates using NB 100-407. Lane 1: HeLa cell lysate, Lanes 2 & 3: bengamide treated lysates (8h and 24h, respectively).

Fig.2: 14-3-3 gamma detected in lysates using NB 100-406. Lane 1: HeLa cell lysates, Lanes 2 and 3: bengamide treated lysates (8h and 24h, respectively).

Fig.3: Detection of 14-3-3 protein in rat hippocampal lysate (showing secificity, lane 1) and lambda-phosphatase incubated rat hippocampal lysate (negative control, lane 2) using NB 100-668.

Fig.4: EB07061 (0.1µg/ml) staining of Human Skin lysate (35µg protein in RIPA buffer). Primary incubation was 1 hour. Detected by chemiluminescence

Fig.5: Formalin-fixed, paraffin-embedded human brain stained with 14.3.3 beta (Cat.#GTX15260) using peroxidase-conjugate and AEC. Note cytoplasmic staining of neurons

Available antibodies to proteins of the 14-3-3 family

14-3-3 pan antibody (YWHA), 14-3-3 beta antibody (YWHAB), 14-3-3 gamma antibody (YWHAG), 14-3-3 epsilon antibody (YWHAE), 14-3-3 zeta antibody (YWHAZ), 14-3-3 theta antibody (YWHAQ), 14-3-3 sigma antibody (stratifin), 14-3-3 eta antibody (YWHAH)

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