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Antibody Sampler Kit Cytokine Production

The ALK Activation Antibody Sampler Kit provides an economical means to evaluate the activation status of multiple members of the ALK pathway, including phosphorylated ALK, Jak2, Jak3, Stat3, Stat5, PLCγ1, Akt, Src, and p44/42 MAPK. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Background: Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor for pleiotrophin (PTN), a growth factor involved in embryonic brain development (1-3). In ALK-expressing cells, PTN induces phosphorylation of both ALK and the downstream effectors IRS-1, Shc, PLCγ, and PI3 kinase (1). ALK was originally discovered as a nucleophosmin (NPM)-ALK fusion protein produced by a translocation (4). Investigators have found that the NPM-ALK fusion protein is a constitutively active, oncogenic tyrosine kinase associated with anaplastic lymphoma (4). Research literature suggests that activation of PLCγ by NPM-ALK may be a crucial step for its mitogenic activity and involved in the pathogenesis of anaplastic lymphomas (5).A distinct ALK oncogenic fusion protein involving ALK and echinoderm microtubule-associated protein like 4 (EML4) has been described in the research literature from a non-small cell lung cancer (NSCLC) cell line, with corresponding fusion transcripts present in some cases of lung adenocarcinoma. The short, amino-terminal region of the microtubule-associated protein EML4 is fused to the kinase domain of ALK (6-8).

The Phospho-p38 MAPK Pathway Sampler Kit provides an economical means to evaluate the activation status of multiple members of the p38 MAPK pathway, including phosphorylated MSK1, p38 MAPK, MKK3/MKK6, ATF-2, HSP27 and MAPKAPK-2. The kit includes enough primary and secondary antibodies to perform two Western blot experiments.

Background: p38 MAP kinase (MAPK), also called RK (1) or CSBP (2), is the mammalian orthologue of the yeast HOG kinase that participates in a signaling cascade controlling cellular responses to cytokines and stress (1-4). Four isoforms of p38 MAPK, p38α, β, γ (also known as Erk6 or SAPK3), and δ (also known as SAPK4) have been identified. Similar to the SAPK/JNK pathway, p38 MAPK is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharide (LPS), UV light, and growth factors (1-5). MKK3, MKK6, and SEK activate p38 MAPK by phosphorylation at Thr180 and Tyr182. Activated p38 MAPK has been shown to phosphorylate and activate MAPKAP kinase 2 (3) and to phosphorylate the transcription factors ATF-2 (5), Max (6), and MEF2 (5-8). SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole) is a selective inhibitor of p38 MAPK. This compound inhibits the activation of MAPKAPK-2 by p38 MAPK and subsequent phosphorylation of HSP27 (9). SB203580 inhibits p38 MAPK catalytic activity by binding to the ATP-binding pocket, but does not inhibit phosphorylation of p38 MAPK by upstream kinases (10).

Senescence Associated Secretory Phenotype (SASP) Antibody Sampler Kit provides an economical means of detecting multiple components of the SASP. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Background: Senescence is characterized by stable stress-induced proliferative arrest and resistance to mitogenic stimuli, as well as the secretion of proteins such as cytokines, growth factors and proteases. These secreted proteins comprise the senescence-associated secretory phenotype (SASP). Senescent cells are thought to accumulate as an organism ages, and contribute to age-related diseases, including cancer, through promotion of inflammation and disruption of normal cellular function (1,2). The composition of the SASP varies, and SASP components can be either beneficial or deleterious in human disease, depending on the context (3).Senescence Associated Secretory Phenotype (SASP) Antibody Sampler Kit provides a collection of antibodies to various SASP components, including TNF-alpha, interleukin-6 (IL-6), the multifunctional cytokine IL-1beta, the chemokines CXCL10, RANTES/CCL5 and MCP-1, the matrix metalloprotease MMP3, and the serine-protease inhibitor PAI-1.

The Toll-Like Receptor Antibody Sampler Kit is an economical way to examine the total protein levels of a number of toll-like receptors. This kit includes enough primary and secondary antibodies to perform two Western blot experiments with each antibody.

Background: Members of the Toll-like receptor (TLR) family, named for the closely related Toll receptor in Drosophila, play a pivotal role in innate immune responses (1-4). TLRs recognize conserved motifs found in various pathogens and mediate defense responses (5-7). Triggering of the TLR pathway leads to the activation of NF-κB and subsequent regulation of immune and inflammatory genes (4). The TLRs and members of the IL-1 receptor family share a conserved stretch of approximately 200 amino acids known as the Toll/Interleukin-1 receptor (TIR) domain (1). Upon activation, TLRs associate with a number of cytoplasmic adaptor proteins containing TIR domains, including myeloid differentiation factor 88 (MyD88), MyD88-adaptor-like/TIR-associated protein (MAL/TIRAP), Toll-receptor-associated activator of interferon (TRIF), and Toll-receptor-associated molecule (TRAM) (8-10). This association leads to the recruitment and activation of IRAK1 and IRAK4, which form a complex with TRAF6 to activate TAK1 and IKK (8,11-14). Activation of IKK leads to the degradation of IκB, which normally maintains NF-κB in an inactive state by sequestering it in the cytoplasm.

The Senescence Marker Antibody Sampler Kit provides an economical means of detecting multiple markers of cellular senescence. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Background: Senescence is characterized by stable stress-induced proliferative arrest and resistance to mitogenic stimuli, as well as the secretion of proteins such as cytokines, growth factors and proteases. These secreted proteins comprise the senescence-associated secretory phenotype (SASP). Senescent cells are thought to accumulate as an organism ages, and contribute to age-related diseases, including cancer, through promotion of inflammation and disruption of normal cellular function (1,2).Because there is no single biomarker that can be used to definitively identify senescent cells, researchers must rely on a collection of biomarkers commonly associated with senescence. The Senescence Marker Antibody Sampler Kit provides a collection of antibodies to commonly used biomarkers of senescence-associated cell cycle arrest (p16 INK4A, p21 Waf1/Cip1), senescence-associated DNA damage (gamma-Histone H2A.X), and the SASP (HMGB1, IL-6, TNF-alpha, MMP3). The kit also includes an antibody to Lamin B1, which is frequently reduced in senescent cells.

The Phospho-Jak Family Antibody Sampler Kit provides an economical means of detecting the activation of Jak family members using phospho-specific and control antibodies. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Background: Members of the Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) are activated by ligands binding to a number of associated cytokine receptors (1). Upon cytokine receptor activation, Jak proteins become autophosphorylated and phosphorylate their associated receptors to provide multiple binding sites for signaling proteins. These associated signaling proteins, such as Stats (2), Shc (3), insulin receptor substrates (4), and focal adhesion kinase (FAK) (5), typically contain SH2 or other phosphotyrosine-binding domains.Activation of Jak kinases upon cytokine receptor binding is associated with tyrosine phosphorylation within their activation loops, including Tyr1034/1035 of Jak1, Tyr1007/1008 of Jak2, Tyr980/981 of Jak3, and Tyr1054/1055 of Tyk2. Many studies have indicated that various cytokine receptors have clear preferences that utilize distinct Jak family members. Aberrant regulation of Jak signaling is associated with a number of diseases, including myeloproliferative neoplasms, leukemia, and inflammatory disease (6).

The Mouse Immune Cell Phenotyping IHC Antibody Sampler Kit provides an economical means of detecting the accumulation of immune cell types in formalin-fixed, paraffin-embedded tissue samples.

Background: Cluster of Differentiation 3 (CD3) is a multiunit protein complex expressed on the surface of T-cells that directly associates with the T-cell receptor (TCR). CD3 is composed of four polypeptides: ζ, γ, ε and δ. Engagement of TCR complex with antigens presented in Major Histocompatibility Complexes (MHC) induces tyrosine phosphorylation in the immunoreceptor tyrosine-based activation motif (ITAM) of CD3 proteins. CD3 phosphorylation is required for downstream signaling through ZAP-70 and p85 subunit of PI-3 kinase, leading to T cell activation, proliferation, and effector functions (1). Cluster of Differentiation 8 (CD8) is a transmembrane glycoprotein expressed primarily on cytotoxic T cells, but has also been described on a subset of dendritic cells in mice (2,3). On T cells, CD8 is a co-receptor for the TCR, and these two distinct structures are required to recognize antigen bound to MHC Class I (2). Cluster of Differentiation 4 (CD4) is expressed on the surface of T helper cells, regulatory T cells, monocytes, macrophages, and dendritic cells, and plays an important role in the development and activation of T cells. On T cells, CD4 is the co-receptor for the TCR, and these two distinct structures recognize antigen bound to MHC Class II. CD8 and CD4 co-receptors ensure specificity of the TCR–antigen interaction, prolong the contact between the T cell and the antigen presenting cell, and recruit the tyrosine kinase Lck, which is essential for T cell activation (2). Granzyme B is a serine protease expressed by CD8+ cytotoxic T lymphocytes and natural killer (NK) cells and is a key component of the immune response to pathogens and transformed cancer cells (4). Forkhead box P3 (FoxP3) is crucial for the development of T cells with immunosuppressive regulatory properties and is a well-established marker for T regulatory cells (Tregs) (5). CD19 is a co-receptor expressed on B cells that amplifies the signaling cascade initiated by the B cell receptor (BCR) to induce activation. It is a biomarker of B lymphocyte development, lymphoma diagnosis, and can be utilized as a target for leukemia immunotherapies (6,7). F4/80 (EMR1) is a heavily glycosylated G-protein-coupled receptor and is a well-established marker for mouse macrophages (8). CD11c (integrin αX, ITGAX) is a transmembrane glycoprotein highly expressed by dendritic cells, and has also been observed on activated NK cells, subsets of B and T cells, monocytes, granulocytes, and some B cell malignancies including hairy cell leukemia (9,10).

The Angiogenesis Antibody Sampler Kit provides an economical means to investigate the angiogenic pathway downstream of VEGFR2. The kit contains enough primary antibody to perform two western blots per primary antibody.
The Src Family Antibody Sampler Kit provides an economical means of evaluating total levels of Src family member proteins. The kit contains enough primary and secondary antibody to perform two western blots with each antibody.

Background: The Src family of protein tyrosine kinases, which includes Src, Lyn, Fyn, Yes, Lck, Blk, and Hck, are important in the regulation of growth and differentiation of eukaryotic cells (1). Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. While phosphorylation at Tyr416 in the activation loop of the kinase domain upregulates enzyme activity, phosphorylation at Tyr527 in the carboxy-terminal tail by Csk renders the enzyme less active (2).

The NF-κB Family Antibody Sampler Kit II provides an economical means of detecting members of the NF-κB family. The kit includes enough antibody to perform two western blots with each primary antibody.

Background: Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).

This kit contains reagents to examine total protein levels of the five NF-κB/Rel family members: p65/RelA, RelB, c-Rel, NF-κB1 (p105/p50) and NF-κB2 (p100/p52).

Background: Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).

The TRAF Antibody Sampler Kit provides an economical means to evaluate endogenous levels of TRAF1, 2, 3, and 6. The kit contains enough primary and secondary antibodies to perform two western mini-blot experiments.

Background: TRAFs (TNF receptor-associated factors) are a family of multifunctional adaptor proteins that bind to surface receptors and recruit additional proteins to form multiprotein signaling complexes capable of promoting cellular responses (1-3). Members of the TRAF family share a common carboxy-terminal "TRAF domain", which mediates interactions with associated proteins; many also contain amino-terminal Zinc/RING finger motifs. The first TRAFs identified, TRAF1 and TRAF2, were found by virtue of their interactions with the cytoplasmic domain of TNF-receptor 2 (TNFRII) (4). The six known TRAFs (TRAF1-6) act as adaptor proteins for a wide range of cell surface receptors and participate in the regulation of cell survival, proliferation, differentiation, and stress responses.

The B Cell Signaling Antibody Sampler Kit provides an economical means to examine key signaling proteins commonly associated with B cell activation. The provided antibodies allow monitoring of both total protein levels and the phosphorylation state. The kit includes enough primary and secondary antibody to perform two western mini-blot experiments.
The c-Kit Antibody Sampler Kit provides a fast and economical means of evaluating levels of c-Kit receptor protein phosphorylated at the specified sites, as well as total c-Kit receptor levels. The kit contains enough primary and secondary antibody to perform two Western blot experiments.

Background: c-Kit is a member of the subfamily of receptor tyrosine kinases that includes PDGF, CSF-1, and FLT3/flk-2 receptors (1,2). It plays a critical role in activation and growth in a number of cell types including hematopoietic stem cells, mast cells, melanocytes, and germ cells (3). Upon binding with its stem cell factor (SCF) ligand, c-Kit undergoes dimerization/oligomerization and autophosphorylation. Activation of c-Kit results in the recruitment and tyrosine phosphorylation of downstream SH2-containing signaling components including PLCγ, the p85 subunit of PI3 kinase, SHP2, and CrkL (4). Molecular lesions that impair the kinase activity of c-Kit are associated with a variety of developmental disorders (5), and mutations that constitutively activate c-Kit can lead to pathogenesis of mastocytosis and gastrointestinal stromal tumors (6). Tyr719 is located in the kinase insert region of the catalytic domain. c-Kit phosphorylated at Tyr719 binds to the p85 subunit of PI3 kinase in vitro and in vivo (7).

The PI3 Kinase Sampler Kit provides an economical means of studying PI3 kinase subunits in cells. The kit contains enough primary and secondary antibodies to perform two Western blot experiments per primary antibody.

Background: Phosphoinositide 3-kinase (PI3K) catalyzes the production of phosphatidylinositol-3,4,5-triphosphate by phosphorylating phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). Growth factors and hormones trigger this phosphorylation event, which in turn coordinates cell growth, cell cycle entry, cell migration, and cell survival (1). PTEN reverses this process, and research studies have shown that the PI3K signaling pathway is constitutively activated in human cancers that have loss of function of PTEN (2). PI3Ks are composed of a catalytic subunit (p110) and a regulatory subunit. Various isoforms of the catalytic subunit (p110α, p110β, p110γ, and p110δ) have been isolated, and the regulatory subunits that associate with p110α, p110β, and p110δ are p85α and p85β (3). In contrast, p110γ associates with a p101 regulatory subunit that is unrelated to p85. Furthermore, p110γ is activated by βγ subunits of heterotrimeric G proteins (4).

The Phospho-Erk1/2 Pathway Sampler Kit provides an economical means of evaluating multiple members of the Erk pathway as well as their activation state. The kit contains enough primary and secondary antibodies to perform two Western blot experiments.

Background: Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.

The B Cell Signaling Antibody Sampler Kit II provides an economical means to examine key signaling proteins commonly associated with B cell activation. The provided antibodies allow monitoring of both total protein levels and the phosphorylation state. The kit includes enough antibody to perform two western blot experiments with each primary antibody.
The Mitochondrial Marker Antibody Sampler Kit provides an economical means to evaluate relevant mitochondial proteins. This kit contains enough primary antibody to perform two western blots per primary.
The Src Antibody Sampler kit provides an economical means of evaluating total Src protein levels and its phosphorylation status. The kit contains enough primary and secondary antibodies to perform two Western blot experiments per primary antibody.

Background: The Src family of protein tyrosine kinases, which includes Src, Lyn, Fyn, Yes, Lck, Blk, and Hck, are important in the regulation of growth and differentiation of eukaryotic cells (1). Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. While phosphorylation at Tyr416 in the activation loop of the kinase domain upregulates enzyme activity, phosphorylation at Tyr527 in the carboxy-terminal tail by Csk renders the enzyme less active (2).

The Exosomal Marker Antibody Sampler Kit provides an economical means to evaluate the presence of exosomal markers. The kit includes enough primary antibody to perform two western blot experiments for each target.