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Monoclonal Antibody Western Blotting Transcription Factor Binding

$260
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: 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, Mouse, Rat

Application Methods: Immunofluorescence (Immunocytochemistry), Western Blotting

Background: The nuclear factor-like 2 (NRF2) transcriptional activator binds antioxidant response elements (ARE) of target gene promoter regions to regulate expression of oxidative stress response genes. Under basal conditions, the NRF2 inhibitor INrf2 (also called KEAP1) binds and retains NRF2 in the cytoplasm where it can be targeted for ubiquitin-mediated degradation (1). Small amounts of constitutive nuclear NRF2 maintain cellular homeostasis through regulation of basal expression of antioxidant response genes. Following oxidative or electrophilic stress, KEAP1 releases NRF2, thereby allowing the activator to translocate to the nucleus and bind to ARE-containing genes (2). The coordinated action of NRF2 and other transcription factors mediates the response to oxidative stress (3). Altered expression of NRF2 is associated with chronic obstructive pulmonary disease (COPD) (4). NRF2 activity in lung cancer cell lines directly correlates with cell proliferation rates, and inhibition of NRF2 expression by siRNA enhances anti-cancer drug-induced apoptosis (5).

$303
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunoprecipitation, Western Blotting

Background: JunB is a basic region, leucine zipper (bZIP) transcription factor belonging to the Jun family that includes c-Jun and JunD. Jun family members homodimerize or heterodimerize with Fos and ATF proteins to form a functional transcription factor AP-1 (activator protein 1), whose activity is regulated by a variety of physiological and pathological stimuli such as growth factors, infections, and stress signals (1-4). While JunB sometimes antagonizes c-Jun transcriptional activity, it may functionally substitute for c-Jun during development in mice (5-7). JunB regulates hematopoietic stem cell number and plays an important role in the pathogenesis of chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) (8,9).

$122
20 µl
$293
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse, Rat

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

Background: Forkhead box protein A2 (FoxA2, also known as hepatocyte nuclear factor 3β or HNF3β) is a transcription factor that plays an important role in hepatocyte function (1). FoxA2/HNF3β is required for the activation of hepatic gluconeogenic gene expression during fasting (1). Together with the PGC-1β coactivator, FoxA2/HNF3β stimulates the expression of genes involved in fatty acid β-oxidation and therefore increases fatty acid metabolism (2). FoxA2/HNF3β, along with PGC-1β, also activates the expression of microsomal triacylglycerol transfer protein (MTP) and promotes VLDL secretion (2). In addition to its roles in metabolic syndromes, FoxA2/HNF3β is essential for development of the endoderm and midline structures in mouse embryos (3-5).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey

Application Methods: Chromatin IP, Immunoprecipitation, Western Blotting

Background: DOT1-like protein (DOT1L), also known as Lysine N-methyltransferase 4 (KMT4), is a histone methyltransferase that functions to mono-, di-, and tri-methylate histone H3 on lysine 79, a histone modification that is associated with active transcription and plays a role in DNA damage response, cell cycle regulation, and embryonic stem cell development (1). DOT1L is required for the initiation and maintenance of mixed lineage leukemia (MLL)-rearranged leukemias, and selective DOT1L inhibitors such as EP2-5676 show remarkable anti-tumor effects in MLL-rearranged leukemias (2,3). Multiple studies have also implicated DOT1L in solid tumor cancers such as breast cancer, where DOT1L induces neoplastic transformation of immortalized breast cancer cells and promotes tumor initiation and growth. Overexpression of DOT1L is associated with poor prognosis in breast cancer, and selective DOT1L inhibitors are able to suppress proliferation and migration of breast cancer cells (4-6).

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

Application Methods: Chromatin IP, Immunoprecipitation, Western Blotting

Background: Max-like protein X (MLX), also known as transcription factor-like protein 4 (TCFL4), is a member of the Myc/Max/Mad network of transcriptional regulator proteins that share a common basic-helix-loop-helix zipper (bHLH-ZIP) motif required for dimerization and DNA-binding (1,2). MLX is ubiquitously expressed in most cell lines and functions as a binding partner for MLXIP (also known as MondoA) and MLXIPL (also known as ChREBP) (1,2). MLX/MLXIP and MLX/MLXIPL heterodimers function to regulate glucose homeostasis by sensing glucose metabolites in the cell. These heterodimeric protein complexes reside mainly in the cytoplasm and mitochondria of cells grown in low glucose, and translocate to the nucleus upon increased intracellular glucose levels to activate transcription of downstream target genes (1,2). MLX/MLXIP is required for the deregulated Myc-induced reprogramming of multiple metabolic pathways during oncogenesis (3).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse, Rat

Application Methods: Western Blotting

Background: The nuclear factor-like 2 (NRF2) transcriptional activator binds antioxidant response elements (ARE) of target gene promoter regions to regulate expression of oxidative stress response genes. Under basal conditions, the NRF2 inhibitor INrf2 (also called KEAP1) binds and retains NRF2 in the cytoplasm where it can be targeted for ubiquitin-mediated degradation (1). Small amounts of constitutive nuclear NRF2 maintain cellular homeostasis through regulation of basal expression of antioxidant response genes. Following oxidative or electrophilic stress, KEAP1 releases NRF2, thereby allowing the activator to translocate to the nucleus and bind to ARE-containing genes (2). The coordinated action of NRF2 and other transcription factors mediates the response to oxidative stress (3). Altered expression of NRF2 is associated with chronic obstructive pulmonary disease (COPD) (4). NRF2 activity in lung cancer cell lines directly correlates with cell proliferation rates, and inhibition of NRF2 expression by siRNA enhances anti-cancer drug-induced apoptosis (5).

$111
20 µl
$260
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse, Rat

Application Methods: Immunoprecipitation, Western Blotting

Background: Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation but has also been associated with a number of physiological processes including development, differentiation, neurodegeneration, infection, and cancer (3). The molecular machinery of autophagy was largely discovered in yeast and referred to as autophagy-related (Atg) genes. Formation of the autophagosome involves a ubiquitin-like conjugation system in which Atg12 is covalently bound to Atg5 and targeted to autophagosome vesicles (4-6). This conjugation reaction is mediated by the ubiquitin E1-like enzyme Atg7 and the E2-like enzyme Atg10 (7,8).

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

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

Background: The CXXC-type zinc finger protein 5 (CXXC5) is a nuclear protein that regulates gene expression and is involved in the regulation of cell growth and differentiation, apoptosis, cell adhesion, and cytoskeletal organization. The CXXC5 protein is also known as retinoid-inducible nuclear factor (RINF) as it was originally identified from a set of genes upregulated by retinoic acid stimulation (1). CXXC5 is a transcriptional activator of the vascular endothelial growth factor receptor VEGFR2. The CXXC5 protein regulates differentiation and migration of endothelial cells and subsequent blood vessel formation downstream of bone morphogenic protein (BMP) signaling (2). CXXC5 also regulates TNFα-induced apoptosis by facilitating phosphorylation of Smad3 and the nuclear translocation of Smad4 (3). Expression of CXXC5 in skeletal muscle regulates expression of genes involved in skeletal myogenesis (4). This nuclear factor also plays an important role in the regulation of normal myelopoiesis. The CXXC5 gene is localized to the 5q31.2 chromosomal region that is often involved in abnormalities associated with various myeloid malignancies, and CXXC5 over-expression is associated with decreased overall survival in human AML (5). Interestingly, CXXC5 is also over-expressed in many solid tumors, and high expression is also correlated with poor prognosis in breast cancer (6).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey

Application Methods: Immunoprecipitation, Western Blotting

Background: The retinoblastoma (Rb) tumor suppressor family includes the retinoblastoma protein Rb (p105), retinoblastoma-like protein 1 (RBL1, p107), and retinoblastoma-like protein 2 (RBL2, p130). These Rb family proteins are referred to as "pocket proteins" because they contain a conserved binding pocket region that interacts with critical regulatory proteins, including E2F family transcription factors, c-Abl tyrosine kinase, and proteins containing a conserved LXCXE motif (1,2). In quiescent G0 phase cells, active Rb proteins hypophosphorylate and bind to E2F transcription factors to repress transcription and inhibit cell cycle progression (1,2). Upon growth factor induction of quiescent cells, Rb proteins become hyperphosphorylated and inactivated by G1-phase cyclinD-cdk4/6, G1/S-phase cyclin E-cdk2, and G1/S-phase cyclin A-cdk2 complexes (1,2). Hyperphosphorylation of Rb proteins results in a loss of E2F binding and allows for transcriptional activation and cell cycle progression (1,2). In addition to regulating the cell cycle, Rb proteins regulate chromosome stability, induction, and maintenance of senescence, apoptosis, cellular differentiation, and angiogenesis (3).Retinoblastoma-like protein 1 (RBL1, p107) interacts with E2F4 and E2F5 to recruit the DP, RB-like, E2F, and MuvB protein (DREAM) complex to E2F target genes to repress transcription of multiple genes required for progression into S phase and mitosis (4-6). Hypophosphorylation of RBL1 during cellular senescence is required for maintenance of senescent cells (7,8).

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

Application Methods: Immunoprecipitation, Western Blotting

Background: DOT1-like protein (DOT1L), also known as Lysine N-methyltransferase 4 (KMT4), is a histone methyltransferase that functions to mono-, di-, and tri-methylate histone H3 on lysine 79, a histone modification that is associated with active transcription and plays a role in DNA damage response, cell cycle regulation, and embryonic stem cell development (1). DOT1L is required for the initiation and maintenance of mixed lineage leukemia (MLL)-rearranged leukemias, and selective DOT1L inhibitors such as EP2-5676 show remarkable anti-tumor effects in MLL-rearranged leukemias (2,3). Multiple studies have also implicated DOT1L in solid tumor cancers such as breast cancer, where DOT1L induces neoplastic transformation of immortalized breast cancer cells and promotes tumor initiation and growth. Overexpression of DOT1L is associated with poor prognosis in breast cancer, and selective DOT1L inhibitors are able to suppress proliferation and migration of breast cancer cells (4-6).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse, Rat

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

Background: The transcription factor proline, glutamic acid, and leucine rich protein 1 (PELP1, MNAR) mediates cell signaling through direct interaction with hormone nuclear receptors to regulate target gene transcription. This versatile protein also regulates gene expression by participating in chromatin remodeling, and acts as a cytoplasmic scaffold protein to mediate growth factor and hormone signaling (1). Following its original description as an estrogen receptor α (ERα) coactivator (2), additional research showed that PELP1 corepresses multiple nuclear hormone receptors and transcriptional regulators, including progesterone receptor, glucocorticoid receptor, AP1, and Stat3 (3). PELP1 also acts cooperatively with the secondary coactivator CARM1 at ERα target gene promoters to increase ERα-mediated transactivation (4). The PELP1 protein contains several leucine-rich repeats, important for interaction with nuclear receptors, and a carboxy-terminal glutamic acid-rich domain responsible for histone protein interaction (2). The glutamic acid-rich region of PELP1 binds to hypoacetylated histones H3 and H4 to block interaction between histone proteins and acetyltransferases. This interaction maintains histones in a hypoacetylated state and suppresses serum-response gene activation. Interaction between PELP1 and ERα relieves this repression and promotes acetylation of histone proteins (3).Research studies demonstrate altered regulation of PELP1 in several distinct hormone-dependent cancers, such as ovarian, breast, and prostate cancers (5-7). As a result, PELP1 may be a promising prognostic marker for hormone-dependent cancers, and inhibiting PELP1 expression or activity may prove beneficial in disrupting hormonal cancer initiation, progression, and metastasis (8).

$111
20 µl
$260
100 µl
APPLICATIONS
REACTIVITY
Human, Mouse, Rat

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

Background: The Fos family of nuclear oncogenes includes c-Fos, FosB, Fos-related antigen 1 (FRA1), and Fos-related antigen 2 (FRA2) (1). While most Fos proteins exist as a single isoform, the FosB protein exists as two isoforms: full-length FosB and a shorter form, FosB2 (Delta FosB), which lacks the carboxy-terminal 101 amino acids (1-3). The expression of Fos proteins is rapidly and transiently induced by a variety of extracellular stimuli including growth factors, cytokines, neurotransmitters, polypeptide hormones, and stress. Fos proteins dimerize with Jun proteins (c-Jun, JunB, and JunD) to form Activator Protein-1 (AP-1), a transcription factor that binds to TRE/AP-1 elements and activates transcription. Fos and Jun proteins contain the leucine-zipper motif that mediates dimerization and an adjacent basic domain that binds to DNA. The various Fos/Jun heterodimers differ in their ability to transactivate AP-1 dependent genes. In addition to increased expression, phosphorylation of Fos proteins by Erk kinases in response to extracellular stimuli may further increase transcriptional activity (4-6). Phosphorylation of c-Fos at Ser32 and Thr232 by Erk5 increases protein stability and nuclear localization (5). Phosphorylation of FRA1 at Ser252 and Ser265 by Erk1/2 increases protein stability and leads to overexpression of FRA1 in cancer cells (6). Following growth factor stimulation, expression of FosB and c-Fos in quiescent fibroblasts is immediate, but very short-lived, with protein levels dissipating after several hours (7). FRA1 and FRA2 expression persists longer, and appreciable levels can be detected in asynchronously growing cells (8). Deregulated expression of c-Fos, FosB, or FRA2 can result in neoplastic cellular transformation; however, Delta FosB lacks the ability to transform cells (2,3).

$260
100 µl
APPLICATIONS
REACTIVITY
Human

Application Methods: Immunohistochemistry (Paraffin), Western Blotting

Background: Human p14 ARF (p19 ARF in mouse) is a pro-apoptotic cell cycle regulator frequently inactive in human tumors (1). Basal expression of p14 ARF is low in most cell types, but accumulation of this protein occurs in response to oncogene expression (2,3). Increased p14 ARF levels facilitate MDM2 degradation, leading to increased p53 protein levels and subsequent cell cycle arrest and/or apoptosis (4). While most p14 ARF signaling has traditionally thought to be p53-dependent, more recent reports have described p53-independent p14 ARF signaling leading to cell cycle arrest and apoptosis (5,6).

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

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

Background: JunB is a basic region, leucine zipper (bZIP) transcription factor belonging to the Jun family that includes c-Jun and JunD. Jun family members homodimerize or heterodimerize with Fos and ATF proteins to form a functional transcription factor AP-1 (activator protein 1), whose activity is regulated by a variety of physiological and pathological stimuli such as growth factors, infections, and stress signals (1-4). While JunB sometimes antagonizes c-Jun transcriptional activity, it may functionally substitute for c-Jun during development in mice (5-7). JunB regulates hematopoietic stem cell number and plays an important role in the pathogenesis of chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) (8,9).

$122
20 µl
$293
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

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

Background: Ubiquitinating enzymes (UBEs) catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) action (1,2). Five DUB subfamilies are recognized, including the USP, UCH, OTU, MJD and JAMM enzymes. Herpesvirus-associated ubiquitin-specific protease (HAUSP, USP7) is an important deubiquitinase belonging to USP subfamily. A key HAUSP function is to bind and deubiquitinate the p53 transcription factor and an associated regulator protein Mdm2, thereby stabilizing both proteins (3,4). In addition to regulating essential components of the p53 pathway, HAUSP also modifies other ubiquitinylated proteins such as members of the FoxO family of forkhead transcription factors and the mitotic stress checkpoint protein CHFR (5,6).

$122
20 µl
$293
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey, Mouse, Rat

Application Methods: Chromatin IP, Immunohistochemistry (Paraffin), Immunoprecipitation, Western Blotting

Background: Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that plays a critical role in the cellular response to hypoxia (1). The HIF1 complex consists of two subunits, HIF-1α and HIF-1β, which are basic helix-loop-helix proteins of the PAS (Per, ARNT, Sim) family (2). HIF1 regulates the transcription of a broad range of genes that facilitate responses to the hypoxic environment, including genes regulating angiogenesis, erythropoiesis, cell cycle, metabolism, and apoptosis. The widely expressed HIF-1α is typically degraded rapidly in normoxic cells by the ubiquitin/proteasomal pathway. Under normoxic conditions, HIF-1α is proline hydroxylated leading to a conformational change that promotes binding to the von Hippel Lindau protein (VHL) E3 ligase complex; ubiquitination and proteasomal degradation follows (3,4). Both hypoxic conditions and chemical hydroxylase inhibitors (such as desferrioxamine and cobalt) inhibit HIF-1α degradation and lead to its stabilization. In addition, HIF-1α can be induced in an oxygen-independent manner by various cytokines through the PI3K-AKT-mTOR pathway (5-7).HIF-1β is also known as AhR nuclear translocator (ARNT) due to its ability to partner with the aryl hydrocarbon receptor (AhR) to form a heterodimeric transcription factor complex (8). Together with AhR, HIF-1β plays an important role in xenobiotics metabolism (8). In addition, a chromosomal translocation leading to a TEL-ARNT fusion protein is associated with acute myeloblastic leukemia (9). Studies also found that ARNT/HIF-1β expression levels decrease significantly in pancreatic islets from patients with type 2 diabetes, suggesting that HIF-1β plays an important role in pancreatic β-cell function (10).

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

Application Methods: Immunoprecipitation, Western Blotting

Background: The process of SUMO conjugation to target proteins is similar to the molecular chain of events observed with ubiquitin (1). SUMO is conjugated to target proteins through the coordinated action of the cellular SUMO conjugation machinery consisting of E1, E2, and E3 enzymes (2). The canonical SUMO E1 activating enzyme is a heterodimer consisting of SAE1 (AOS1) and UBA2 (SAE2) subunits. Mature SUMO is activated by E1 in an ATP-dependent reaction that generates adenylated SUMO, which functions as a high-energy intermediate in the formation of a thioester linkage between SUMO and Cys173 of UBA2 (3,4). SUMO is subsequently transferred from UBA2 to the SUMO E2 conjugating enzyme, UBC9 (5). Recent evidence suggests that redox regulation of UBA2 serves as a physiologic mechanism to modulate the cellular level of sumoylated target proteins (6).

$260
100 µl
APPLICATIONS
REACTIVITY
Human

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

Background: Inhibitor of DNA-binding/Differentiation (ID) proteins are a family of proteins that function to repress the activity of basic helix-loop-helix (bHLH) transcription factors. There are four known ID proteins in humans (ID1-4), all of which contain a helix-loop-helix domain but lack a basic DNA binding domain. Heterodimerization with bHLH transcription factors therefore functions to sequester bHLH proteins and prevent their binding to DNA (1). ID proteins play important functional roles in development, primarily by inhibiting premature differentiation of stem/progenitor cells (1,2). ID3 plays an important role in immune system development where it has been shown to repress E2A-mediated differentiation of T cells (3). Studies in mouse models have shown that homozygous deletion of ID3 disrupts regulatory T cell differentiation (4) and leads to development of γδ T cell lymphoma (5). Outside of the hematopoietic compartment, ID3 was shown to repress MyoD, implicating ID3 in TGFβ-mediated muscle repair (6). Similarly, research studies have shown that ID3 suppresses p21 in colon cancer cells, a function that is purported to promote the self-renewal capacity of putative cancer-initiating cells (7).

$260
100 µl
APPLICATIONS
REACTIVITY
Human, Monkey

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

Background: DNA-dependent protein kinase (DNA-PK) is an important factor in the repair of double-stranded breaks in DNA. Cells lacking DNA-PK or in which DNA-PK is inhibited fail to show proper nonhomologous end-joining (NHEJ) (1-7). DNA-PK is composed of two DNA-binding subunits (Ku70 and Ku86) and one 450 kDa catalytic subunit (DNA-PKcs) (8). It is thought that a heterodimer of Ku70 and Ku86 binds to double-stranded DNA broken ends before DNA-PKcs binds and is activated (1,9). Activated DNA-PKcs is a serine/threonine kinase that has been shown to phosphorylate a number of proteins in vitro, including p53, transcription factors, RNA polymerase, and Ku70/Ku86 (10,11). DNA-PKcs autophosphorylation at multiple sites, including Thr2609 and Ser2056, results in an inactivation of DNA-PK kinase activity and NHEJ ability (12,13). It has been demonstrated, however, that DNA-PK preferentially phosphorylates substrates before it autophosphorylates, suggesting that DNA-PK autophosphorylation may play a role in disassembly of the DNA repair machinery (14,15). Autophosphorylation at Thr2609 has also been shown to be required for DNA-PK-mediated double strand break repair, and phosphorylated DNA-PK co-localizes with H2A.X and 53BP1 at sites of DNA damage (16). Phosphorylation at Ser2056 occurs in response to double-stranded DNA breaks and ATM activation (17).