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Polyclonal Antibody Immunofluorescence Immunocytochemistry Chromatin Dna Binding

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat, Zebrafish

Application Methods: Immunofluorescence (Immunocytochemistry), Immunoprecipitation, Western Blotting

Background: Modulation of chromatin structure plays a critical role in the regulation of transcription in eukaryotes. The nucleosome, made up of four core histone proteins (H2A, H2B, H3 and H4), is the primary building block of chromatin. In addition to the growing number of post-translational histone modifications regulating chromatin structure, cells can also exchange canonical histones with variant histones that can directly or indirectly modulate chromatin structure (1). There are five major variants of histone H2A: canonical H2A (most abundant), H2A.X, MacroH2A, H2ABbd and H2A.Z (2). Histone H2A.Z, the most conserved variant across species, functions as both a positive and negative regulator of transcription and is important for chromosome stability (2). Several homologous protein complexes, such as SWR-C (S. cerevisiae), TIP60 (D. melanogaster) and SRCAP (mammals), have been shown to catalyze the ATP-dependent exchange of H2A.Z for H2A in the nucleosome (3,4,5). This exchange of histone H2A variants changes histone-histone interactions in the nucleosome core and alters an acidic patch on the surface of the nucleosome, resulting in changes in nucleosome stability and binding of non-histone proteins such as HP1α (6,7).

$303
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Immunohistochemistry (Paraffin), Immunoprecipitation, Western Blotting

Background: Acetylation of the histone tail causes chromatin to adopt an "open" conformation, allowing increased accessibility of transcription factors to DNA. The identification of histone acetyltransferases (HATs) and their large multiprotein complexes has yielded important insights into how these enzymes regulate transcription (1,2). HAT complexes interact with sequence-specific activator proteins to target specific genes. In addition to histones, HATs can acetylate nonhistone proteins, suggesting multiple roles for these enzymes (3). In contrast, histone deacetylation promotes a "closed" chromatin conformation and typically leads to repression of gene activity (4). Mammalian histone deacetylases can be divided into three classes on the basis of their similarity to various yeast deacetylases (5). Class I proteins (HDACs 1, 2, 3, and 8) are related to the yeast Rpd3-like proteins, those in class II (HDACs 4, 5, 6, 7, 9, and 10) are related to yeast Hda1-like proteins, and class III proteins are related to the yeast protein Sir2. Inhibitors of HDAC activity are now being explored as potential therapeutic cancer agents (6,7).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: Histone macroH2A1 and macroH2A2 comprise a family of variant histone H2A proteins. MacroH2A1 exists as two distinct isoforms due to alternative splicing of a single gene; macroH2A1.1 levels accumulate throughout differentiation and development while macroH2A1.2 shows a constant level of expression (1). MacroH2A1 and macroH2A2 are encoded by completely distinct genes located on separate chromosomes (2,3). Both macroH2A1 and macroH2A2 proteins contain an amino-terminal histone-like region with 64% sequence identity to canonical histone H2A, in addition to a carboxy-terminal “macro” domain (1-3). MacroH2A1 and macroH2A2 are enriched in facultative heterochromatin, including inactivated X chromosomes in mammalian females and senescence-associated heterochromatin foci (2-5). Both act to repress gene transcription by inhibiting the binding of transcription factors to chromatin, the acetylation of histones by p300, and the chromatin-remodeling activities of SWI/SNF and ACF (6,7). The macro domain of macroH2A1.1 binds to ADP-ribose and functions to recruit macroH2A1.1 to activated PARP at sites of DNA damage, where it mediates chromatin rearrangements to locally regulate the DNA damage response (8). MacroH2A1.2 and macroH2A2 do not bind poly-ADP-ribose and are not recruited to sites of activated PARP (8).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Chromatin IP, Flow Cytometry, Immunofluorescence (Immunocytochemistry), Immunoprecipitation, Western Blotting

Background: Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, and 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Immunoprecipitation, Western Blotting

Background: Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, and 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).

$303
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunofluorescence (Immunocytochemistry), Immunoprecipitation, Western Blotting

Background: Modulation of chromatin structure plays a critical role in the regulation of transcription and replication of the eukaryotic genome. The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. In addition to the growing number of post-translational histone modifications regulating chromatin structure, cells can also exchange canonical histones with variant histones that can directly or indirectly modulate chromatin structure (1). CENP-A, also known as the chromatin-associated protein CSE4 (capping-enzyme suppressor 4-p), is an essential histone H3 variant that replaces canonical histone H3 in centromeric heterochromatin (2). The greatest divergence between CENP-A and canonical histone H3 occurs in the amino-terminal tail of the protein, which binds linker DNA between nucleosomes and facilitates proper folding of centromeric heterochromatin (3). The amino-terminal tail of CENP-A is also required for recruitment of other centromeric proteins (CENP-C, hSMC1, hZW10), proper kinetochore assembly and chromosome segregation during mitosis (4). Additional sequence divergence in the histone fold domain is responsible for correct targeting of CENP-A to the centromere (5). Many of the functions of CENP-A are regulated by phosphorylation (6,7). Aurora A-dependent phosphorylation of CENP-A on Ser7 during prophase is required for proper targeting of Aurora B to the inner centromere in prometaphase, proper kinetochore/microtubule attachment and proper alignment of chromosomes during mitosis (6).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: Acetylation of the histone tail causes chromatin to adopt an "open" conformation, allowing increased accessibility of transcription factors to DNA. The identification of histone acetyltransferases (HATs) and their large multiprotein complexes has yielded important insights into how these enzymes regulate transcription (1,2). HAT complexes interact with sequence-specific activator proteins to target specific genes. In addition to histones, HATs can acetylate nonhistone proteins, suggesting multiple roles for these enzymes (3). In contrast, histone deacetylation promotes a "closed" chromatin conformation and typically leads to repression of gene activity (4). Mammalian histone deacetylases can be divided into three classes on the basis of their similarity to various yeast deacetylases (5). Class I proteins (HDACs 1, 2, 3, and 8) are related to the yeast Rpd3-like proteins, those in class II (HDACs 4, 5, 6, 7, 9, and 10) are related to yeast Hda1-like proteins, and class III proteins are related to the yeast protein Sir2. Inhibitors of HDAC activity are now being explored as potential therapeutic cancer agents (6,7).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Chromatin IP, Immunofluorescence (Immunocytochemistry), Immunoprecipitation, Western Blotting

Background: CCCTC-binding factor (CTCF) and its paralog, the Brother of the Regulator of Imprinted Sites (BORIS), are highly conserved transcription factors that regulate transcriptional activation and repression, insulator function, and imprinting control regions (ICRs) (1-4). Although they have divergent amino and carboxy termini, both proteins contain 11 conserved zinc finger domains that work in combination to bind the same DNA elements (1). CTCF is ubiquitously expressed and contributes to transcriptional regulation of cell-growth regulated genes, including c-myc, p19/ARF, p16/INK4A, BRCA1, p53, p27, E2F1, and TERT (1). CTCF also binds to and is required for the enhancer-blocking activity of all known insulator elements and ICRs, including the H19/IgF2, Prader-Willi/Angelman syndrome, and Inactive X-Specific Transcript (XIST) anti-sense loci (5-7). CTCF DNA-binding is sensitive to DNA methylation, a mark that determines selection of the imprinted allele (maternal vs. paternal) (1). The various functions of CTCF are regulated by at least two different post-translational modifications. Poly(ADP-ribosyl)ation of CTCF is required for insulator function (8). Phosphorylation of Ser612 by protein kinase CK2 facilitates a switch of CTCF from a transcriptional repressor to an activator at the c-myc promoter (9). CTCF mutations or deletions have been found in many breast, prostate, and Wilms tumors (10,11). Expression of BORIS is restricted to spermatocytes and is mutually exclusive of CTCF (3). In cells expressing BORIS, promoters of X-linked cancer-testis antigens like MAGE-1A are demethylated and activated, but methylated and inactive in CTCF-expressing somatic cells (12). Like other testis specific proteins, BORIS is abnormally expressed in different cancers, such as breast cancer, and has a greater affinity than CTCF for DNA binding sites, detracting from CTCF’s potential tumor suppressing activity (1,3,13,14).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: High mobility group protein B1 (HMGB1) belongs to a family of highly conserved proteins that contain HMG box domains (1,2). All three family members (HMGB1, HMGB2, and HMGB3) contain two HMG box domains and a C-terminal acidic domain. HMGB1 is a widely expressed and highly abundant protein (2). HMGB2 is widely expressed during embryonic development, but is restricted to lymphoid organs and testis in adult animals (3). HMGB3 is only expressed during embryogenesis (4). While expression varies, the biochemical properties of the different family members may be indistinguishable. The HMG box domains facilitate the binding of HMGB proteins to the minor groove of DNA, which results in local bending of the DNA double helix (1,2). HMGB proteins are recruited by and help facilitate the assembly of site-specific DNA binding proteins to their cognate binding sites in chromatin. For example, HMGB1 facilitates the binding of Hox proteins, Oct-1, p53, Rel proteins, and steroid hormone receptor proteins to their target gene promoters (1,2). In addition to their functions in the nucleus, HMGB proteins play a significant role in extracellular signaling associated with inflammation (5,6). HMGB1 is massively released into the extracellular environment during cell necrosis, but not apoptosis. Extracellular HMGB1 "alarms" the innate immune system by acting as a chemoattractant for inflammatory leukocytes, smooth muscle cells, and stem cells, functioning as an immune adjuvant for soluble and particulate antigens, and triggering activation of T cells and dendritic cells. In addition, activated monocytes, macrophages and, dendritic cells also secrete HMGB1, forming a positive feedback loop that results in the release of additional cytokines and neutrophils. Hypoxia has also been shown to cause the release of HMGB1 in the liver, and some studies suggest a role for extracellular HMGB1 in tumor homeostasis (5,6).

$260
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis (1). In addition to p53, mammalian cells contain two p53 family members, p63 and p73, which are similar to p53 in both structure and function (2). While p63 can induce p53-responsive genes and apoptosis, mutation of p63 rarely results in tumors (2). Research investigators frequently observe amplification of the p63 gene in squamous cell carcinomas of the lung, head and neck (2,3). The p63 gene contains an alternative transcription initiation site that yields a truncated ΔNp63 lacking the transactivation domain, and alternative splicing at the carboxy-terminus yields the α, β, and γ isoforms (3,4).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: The Ras family small GTPase Ran is involved in nuclear envelope formation, assembly of the mitotic spindle, and nuclear transport (1,2). Like other small GTPases, Ran is active in its GTP-bound form and inactive in its GDP-bound form. Nuclear RanGTP concentration is maintained through nuclear localization of guanine nucleotide exchange factor (GEF) activity, which catalyzes the exchange of bound GDP for GTP. Regulator of chromatin condensation 1 (RCC1) is the only known RanGEF (3). RCC1 is dynamically chromatin-bound throughout the cell cycle, and this localization is required for mitosis to proceed normally (4,5). Appropriate association of RCC1 with chromatin is regulated through amino-terminal phosphorylation (5,6) and methylation (7). RCC1 regulation of RanGTP levels in response to histone modifications regulates nuclear import during apoptosis (8). In mitosis RCC1 is phosphorylated at Ser11, possibly by cyclin B/cdc2 (9-11). This phosphorylation may play a role in RCC1 interaction with chromatin and RCC1 RanGEF activity (6).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse

Application Methods: Immunofluorescence (Immunocytochemistry), Immunoprecipitation, Western Blotting

Background: The NFAT (nuclear factor of activated T cells) family of proteins consists of NFAT1 (NFATc2 or NFATp), NFAT2 (NFATc1 or NFATc), NFAT3 (NFATc4), and NFAT4 (NFATc3 or NFATx). All members of this family are transcription factors with a Rel homology domain and regulate gene transcription in concert with AP-1 (Jun/Fos) to orchestrate an effective immune response (1,2). NFAT proteins are predominantly expressed in cells of the immune system, but are also expressed in skeletal muscle, keratinocytes, and adipocytes, regulating cell differentiation programs in these cells (3). In resting cells, NFAT proteins are heavily phosphorylated and localized in the cytoplasm. Increased intracellular calcium concentrations activate the calcium/calmodulin-dependent serine phosphatase calcineurin, which dephosphorylates NFAT proteins, resulting in their subsequent translocation to the nucleus (2). Termination of NFAT signaling occurs upon declining calcium concentrations and phosphorylation of NFAT by kinases such as GSK-3 or CK1 (3,4). Cyclosporin A and FK506 are immunosuppressive drugs that inhibit calcineurin and thus retain NFAT proteins in the cytoplasm (5).

$303
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Flow Cytometry, Immunofluorescence (Immunocytochemistry), Immunohistochemistry (Paraffin), Immunoprecipitation, Western Blotting

Background: The transcription factor ATF-2 (also called CRE-BP1) binds to both AP-1 and CRE DNA response elements and is a member of the ATF/CREB family of leucine zipper proteins (1). ATF-2 interacts with a variety of viral oncoproteins and cellular tumor suppressors and is a target of the SAPK/JNK and p38 MAP kinase signaling pathways (2-4). Various forms of cellular stress, including genotoxic agents, inflammatory cytokines, and UV irradiation, stimulate the transcriptional activity of ATF-2. Cellular stress activates ATF-2 by phosphorylation of Thr69 and Thr71 (2-4). Both SAPK and p38 MAPK have been shown to phosphorylate ATF-2 at these sites in vitro and in cells transfected with ATF-2. Mutations of these sites result in the loss of stress-induced transcription by ATF-2 (2-4). In addition, mutations at these sites reduce the ability of E1A and Rb to stimulate gene expression via ATF-2 (2).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey

Application Methods: Flow Cytometry, Immunofluorescence (Immunocytochemistry), Immunoprecipitation, Western Blotting

Background: RPA70 (HSSB, REPA1, RF-A, RP-A, p70) is a component of a heterotrimeric complex, composed of 70, 32/30 and 14 kDa subunits, collectively known as RPA. RPA is a single stranded DNA binding protein, whose DNA binding activity is believed to reside entirely in the 70 kDa subunit. The complex is required for almost all aspects of cellular DNA metabolism such as DNA replication (1-3), recombination, cell cycle and DNA damage checkpoints, and all major types of DNA repair including nucleotide excision, base excision, mismatch and double-strand break repairs (4-7). In response to genotoxic stress in eukaryotic cells, RPA has been shown to associate with the Rad9/Rad1/Hus1 (9-1-1) checkpoint complex (8). RPA is hyperphosphorylated upon DNA damage or replication stress by checkpoint kinases including ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) (9-11). Phosphorylation of RPA32 occurs at serines 4, 8 and 33 (11). Hyperphosphorylation may alter RPA-DNA and RPA-protein interactions. In addition to the checkpoint partners, RPA interacts with a wide variety of protein partners, including proteins required for normal replication such as RCF, PCNA and Pol α, and also proteins involved in SV40 replication, such as DNA polymerase I and SV40 large T antigen (10,12).

$260
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: Transforming growth factor-β (TGF-β) superfamily members are critical regulators of cell proliferation and differentiation, developmental patterning and morphogenesis and disease pathogenesis (1-3). Upon stimulation by TGF-β, activated receptors phosphorylate Smad2 and Smad3, resulting in their translocation to the nucleus, association with Smad4 and transcriptional regulation of target genes (4). Ski and SnoN are related oncoproteins originally discovered based on homology to v-Ski, the transforming protein of the Sloan-Kettering virus (5). They regulate TGF-β signaling by binding to Smad2 and Smad4 and repressing their ability to activate transcription (6). Following TGF-β stimulation, SnoN is rapidly degraded by the ubiquitin proteasome pathway providing negative feedback regulation (6-9). Overexpression of SnoN and Ski can transform avian fibroblasts and induce muscle differentiation (10). Mice heterozygous for SnoN and Ski display increased susceptibility to tumorigenesis (11,12). Interestingly, elevated expression of Ski and SnoN has been observed in many tumors and may serve as important prognostic markers (13,14). Taken together, these studies suggest possible dual functions of these proteins at different stages of tumorigenesis (15).

$303
100 µl
$717
300 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: c-Jun is a member of the Jun family containing c-Jun, JunB, and JunD, and is a component of the transcription factor activator protein-1 (AP-1). AP-1 is composed of dimers of Fos, Jun, and ATF family members and binds to and activates transcription at TRE/AP-1 elements (reviewed in 1). Extracellular signals including growth factors, chemokines, and stress activate AP-1-dependent transcription. The transcriptional activity of c-Jun is regulated by phosphorylation at Ser63 and Ser73 through SAPK/JNK (reviewed in 2). Knock-out studies in mice have shown that c-Jun is essential for embryogenesis (3), and subsequent studies have demonstrated roles for c-Jun in various tissues and developmental processes including axon regeneration (4), liver regeneration (5), and T cell development (6). AP-1 regulated genes exert diverse biological functions including cell proliferation, differentiation, and apoptosis, as well as transformation, invasion and metastasis, depending on cell type and context (7-9). Other target genes regulate survival, as well as hypoxia and angiogenesis (8,10). Research studies have implicated c-Jun as a promising therapeutic target for cancer, vascular remodeling, acute inflammation, and rheumatoid arthritis (11,12).

$122
20 µl
$303
100 µl
APPLICATIONS
REACTIVITY
Human, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: The epidermal growth factor (EGF) receptor is a transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling, internalization, and lysosomal degradation (1,2). Phosphorylation of EGF receptor (EGFR) at Tyr845 in the kinase domain is implicated in stabilizing the activation loop, maintaining the active state enzyme, and providing a binding surface for substrate proteins (3,4). c-Src is involved in phosphorylation of EGFR at Tyr845 (5). The SH2 domain of PLCγ binds at phospho-Tyr992, resulting in activation of PLCγ-mediated downstream signaling (6). Phosphorylation of EGFR at Tyr1045 creates a major docking site for the adaptor protein c-Cbl, leading to receptor ubiquitination and degradation following EGFR activation (7,8). The GRB2 adaptor protein binds activated EGFR at phospho-Tyr1068 (9). A pair of phosphorylated EGFR residues (Tyr1148 and Tyr1173) provide a docking site for the Shc scaffold protein, with both sites involved in MAP kinase signaling activation (2). Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity. EGFR carboxy-terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II; mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation (10).

$122
20 µl
$303
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey

Application Methods: Flow Cytometry, Immunofluorescence (Immunocytochemistry), Immunoprecipitation, Western Blotting

Background: The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis (1). p53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro (2,3). DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2 (4). MDM2 inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation (5,6). p53 can be phosphorylated by ATM, ATR, and DNA-PK at Ser15 and Ser37. Phosphorylation impairs the ability of MDM2 to bind p53, promoting both the accumulation and activation of p53 in response to DNA damage (4,7). Chk2 and Chk1 can phosphorylate p53 at Ser20, enhancing its tetramerization, stability, and activity (8,9). p53 is phosphorylated at Ser392 in vivo (10,11) and by CAK in vitro (11). Phosphorylation of p53 at Ser392 is increased in human tumors (12) and has been reported to influence the growth suppressor function, DNA binding, and transcriptional activation of p53 (10,13,14). p53 is phosphorylated at Ser6 and Ser9 by CK1δ and CK1ε both in vitro and in vivo (13,15). Phosphorylation of p53 at Ser46 regulates the ability of p53 to induce apoptosis (16). Acetylation of p53 is mediated by p300 and CBP acetyltransferases. Inhibition of deacetylation suppressing MDM2 from recruiting HDAC1 complex by p19 (ARF) stabilizes p53. Acetylation appears to play a positive role in the accumulation of p53 protein in stress response (17). Following DNA damage, human p53 becomes acetylated at Lys382 (Lys379 in mouse) in vivo to enhance p53-DNA binding (18). Deacetylation of p53 occurs through interaction with the SIRT1 protein, a deacetylase that may be involved in cellular aging and the DNA damage response (19).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Immunohistochemistry (Paraffin), Western Blotting

Background: The Y-box binding protein 1 (YB1) belongs to a family of evolutionarily conserved, multifunctional Y-box proteins that bind single-stranded DNA and RNA and function as regulators of transcription, RNA metabolism, and protein synthesis (1). YB1 binds to Y-box sequences (TAACC) found in multiple gene promoters and can positively or negatively regulate transcription. YB1 activates genes associated with proliferation and cancer, such as cyclin A, cyclin B1, matrix metalloproteinase-2 (MMP-2), and the multi-drug resistance 1 (MDR1) gene (2-4). YB1 represses genes associated with cell death, including the Fas cell death-associated receptor and the p53 tumor suppressor gene (5-7). It also interacts with the RNA-splicing factor SRp30c and stabilizes interleukin-2 (IL-2) mRNA upon induction of T lymphocytes by IL-2 (8,9). The majority of YB1 protein localizes to the cytoplasm, with a minor pool found in the nucleus; however, nuclear localization appears to be critical for its role in promoting proliferation. Nuclear translocation is cell cycle regulated, with YB1 protein accumulating in the nucleus during G1/S phase (2). In addition, nuclear translocation is induced in response to extracellular stimuli such as hyperthermia and UV irradiation, or treatment of cells with thrombin, interferons, or insulin-like growth factor (IGF-I) (2,10). Treatment of the MCF7 breast cancer cell line with IGF-I results in Akt-mediated phosphorylation of YB1 at Ser102, which is required for nuclear translocation of YB1 and its ability to promote anchorage-independent growth (10). Research studies have shown that YB1 is overexpressed in many malignant tissues, including breast cancer, non-small cell lung carcinoma, ovarian adenocarcinomas, human osteosarcomas, colorectal carcinomas, and malignant melanomas. Investigators have shown that nuclear YB1 expression correlates with high levels of proliferation, drug resistance, and poor tumor prognosis (2,7,10).