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Antibody Sampler Kit Regulation of Phagocytosis

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 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 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 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 Integrin Antibody Sampler Kit provides an economical means to screen samples for α and β subunits of integrin molecules. The kit includes enough primary and secondary antibody to perform two Western blot experiments with each antibody.
The Adipogenesis Marker Antibody Sampler Kit provides an economical means to evaluate proteins involved in the regulation of adipogenesis. The kit includes enough antibody to perform two western blot experiments with each primary antibody.
The Inflammasome Antibody Sampler Kit provides an economical means of detecting multiple inflammasome components. The kit contains enough primary antibodies to perform at least two western blot experiments.

Background: The innate immune system works as the first line of defense in protection from pathogenic microbes and host-derived signals of cellular distress. One way in which these “danger” signals trigger inflammation is through activation of inflammasomes, which are multiprotein complexes that assemble in the cytosol after exposure to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) and result in the activation of caspase-1 and subsequent cleavage of proinflammatory cytokines IL-1β and IL-18 (Reviewed in 1-6). Inflammasome complexes typically consist of a cytosolic pattern recognition receptor (PRR; a nucleotide-binding domain and leucine-rich-repeat [NLR] or AIM2-like receptor [ALR] family member), an adaptor protein (ASC/TMS1), and pro-caspase-1. A number of distinct inflammasome complexes have been identified, each with a unique PRR and activation triggers. The best characterized is the NLRP3 complex, which contains NLRP3, ASC/TMS1, and pro-caspase-1. The NLRP3 inflammasome is activated in a two-step process. First, NF-κB signaling is induced through PAMP- or DAMP-mediated activation of TLR4 or TNFR, resulting in increased expression of NLRP3, pro-IL-1β, and pro-IL-18 (priming step, signal 1). Next, indirect activation of NLRP3 occurs by a multitude of signals (whole pathogens, PAMPs/DAMPs, potassium efflux, lysosomal-damaging environmental factors [uric acid, silica, alum] and endogenous factors [amyloid-β, cholesterol crystals], and mitochondrial damage), leading to complex assembly and activation of caspase-1 (signal 2). The complex inflammasome structure is built via domain interactions among the protein components. Other inflammasomes are activated by more direct means: double-stranded DNA activates the AIM2 complex, anthrax toxin activates NLRP1, and bacterial flagellin activates NLRC4. Activated caspase-1 induces secretion of proinflammatory cytokines IL-1β and -18, but also regulates metabolic enzyme expression, phagosome maturation, vasodilation, and pyroptosis, an inflammatory programmed cell death. Inflammasome signaling contributes to the onset of a number of diseases, including atherosclerosis, type II diabetes, Alzheimer’s disease, and autoimmune disorders.

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 Human Reactive Inflammasome Antibody Sampler Kit II provides an economical means of detecting multiple inflammasome components. The kit contains enough primary antibodies to perform at least two western blot experiments.

Background: The innate immune system works as the first line of defense in protection from pathogenic microbes and host-derived signals of cellular distress. One way in which these “danger” signals trigger inflammation is through activation of inflammasomes, which are multiprotein complexes that assemble in the cytosol after exposure to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) and result in the activation of caspase-1 and subsequent cleavage of proinflammatory cytokines IL-1β and IL-18 (Reviewed in 1-6). Inflammasome complexes typically consist of a cytosolic pattern recognition receptor (PRR; a nucleotide-binding domain and leucine-rich-repeat [NLR] or AIM2-like receptor [ALR] family member), an adaptor protein (ASC/TMS1), and pro-caspase-1. A number of distinct inflammasome complexes have been identified, each with a unique PRR and activation triggers. The best characterized is the NLRP3 complex, which contains NLRP3, ASC/TMS1, and pro-caspase-1. The NLRP3 inflammasome is activated in a two-step process. First, NF-κB signaling is induced through PAMP- or DAMP-mediated activation of TLR4 or TNFR, resulting in increased expression of NLRP3, pro-IL-1β, and pro-IL-18 (priming step, signal 1). Next, indirect activation of NLRP3 occurs by a multitude of signals (whole pathogens, PAMPs/DAMPs, potassium efflux, lysosomal-damaging environmental factors [uric acid, silica, alum] and endogenous factors [amyloid-β, cholesterol crystals], and mitochondrial damage), leading to complex assembly and activation of caspase-1 (signal 2). The complex inflammasome structure is built via domain interactions among the protein components. Other inflammasomes are activated by more direct means: double-stranded DNA activates the AIM2 complex, anthrax toxin activates NLRP1, and bacterial flagellin activates NLRC4. Activated caspase-1 induces secretion of proinflammatory cytokines IL-1β and -18, but also regulates metabolic enzyme expression, phagosome maturation, vasodilation, and pyroptosis, an inflammatory programmed cell death. Inflammasome signaling contributes to the onset of a number of diseases, including atherosclerosis, type II diabetes, Alzheimer’s disease, and autoimmune disorders.

The Microglia Cross Module Antibody Sampler Kit provides an economical means of detecting proteins identified as markers of microglial activity corresponding to proliferation, neurodegeneration, interferon and LPS-relation by western blot and/or immunofluorescence.

Background: Distinct microglial activation states have been identified using RNA-seq data from a vast array of neurological disease and aging models. These activation states have been categorized into modules corresponding to proliferation, neurodegeneration, interferon-relation, LPS-relation, and many others (1). Previous work identifying markers of specific brain cell types using RNA-seq has shown HS1 and ASC/TMS1 to be useful and specific tools to study microglia (2). HS1 is a protein kinase substrate that is expressed only in tissues and cells of hematopoietic origin (3) and ASC/TMS1 has been found to be a critical component of inflammatory signaling where it associates with and activates caspase-1 in response to pro-inflammatory signals (4).

The Microglia Neurodegeneration Module Antibody Sampler Kit provides an economical means of detecting proteins identified as markers of microglial activity during neurodegeneration by western blot and/or immunofluorescence.

Background: Distinct microglial activation states have been identified using RNA-seq data from a vast array of neurological disease and aging models. These activation states have been categorized into modules corresponding to proliferation, neurodegeneration, interferon-relation, LPS-relation, and many others (1). Previous work identifying markers of specific brain cell types using RNA-seq has shown HS1 and ASC/TMS1 to be useful and specific tools to study microglia (2). HS1 is a protein kinase substrate that is expressed only in tissues and cells of hematopoietic origin (3) and ASC/TMS1 has been found to be a critical component of inflammatory signaling where it associates with and activates caspase-1 in response to pro-inflammatory signals (4).

Background: Microglia cells are resident macrophages of the brain that survey the brain environment and dynamically respond to maintain brain homeostasis. Microglial responses include phagocytosis of cellular debris, restricting sites injury or pathology, and/or releasing inflammatory signals to initiate an immune response. Such responses are important during normal development and during diseased states (1).Recently, the role of microglia in neurodegenerative disease pathology, particularly Alzheimer’s disease (AD), has been of intense investigation. Much of this work is driven by human genetic data that links microglia-enriched genes with AD progression (2). The triggering receptor expressed on myeloid cells 2 (TREM2) protein is an innate immune receptor that is expressed on the cell surface of microglia (3). TREM2 plays a role in innate immunity, and a rare functional variant (R47H) of the TREM2 gene is associated with the late-onset risk of AD (3,4). How TREM2 contributes to disease function is currently an active area of research (4,5), but might drive a number of microglial cellular functions ranging from microgliosis, phagocytosis, and cytokine release via a variety of signaling cascades triggered by TREM2.The TREM2 receptor is a single-pass type I membrane glycoprotein that consists of an extracellular immunoglobulin-like domain, a transmembrane domain, and a cytoplasmic tail. Ligands for TREM2 include phospholipids, apolipoproteins, and lipoproteins. Upon activation, TREM2 interacts with the tyrosine kinase-binding protein DNAX-activating protein 12 (DAP12, TYROBP) to form a receptor-signaling complex (6). Ligand binding by DAP12-associated receptors, including TREM2, results in phosphorylation of tyrosine residues within the DAP12 immunoreceptor tyrosine-based activation motif (ITAM) by Src family kinases; ITAM phosphorylation leads to activation of spleen tyrosine kinase (Syk) and downstream signaling cascades (7). Tyr525 and Tyr526 are located in the activation loop of the Syk kinase domain and phosphorylation at these residues (equivalent to Tyr519/520 of mouse Syk) is essential for Syk function (8). Syk phosphorylation is also a readout for β-amyloid triggered TREM2 activity (9). Phosphoinositide-specific phospholipase C γ 1/2 (PLCγ1/2) is reported to be down stream of Syk (10). Tyr352 of Syk is involved in the association of PLCγ1 (11); Syk-mediated phosphorylation PLCγ1 at Tyr783 activates PLCγ1 enzymatic activity (12). Interestingly, mutations in the microglia-enriched PLCγ2 gene are associated with AD (13,14,15).

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 PTEN and PDK1 Sampler Kit provides an economical means to evaluate two key enzymes that regulate multiple signaling pathways. The kit contains enough primary and secondary antibodies to perform two Western blots per primary antibody.

Background: PTEN (phosphatase and tensin homologue deleted on chromosome ten), also referred to as MMAC (mutated in multiple advanced cancers) phosphatase, is a tumor suppressor implicated in a wide variety of human cancers (1). PTEN encodes a 403 amino acid polypeptide originally described as a dual-specificity protein phosphatase (2). The main substrates of PTEN are inositol phospholipids generated by the activation of the phosphoinositide 3-kinase (PI3K) (3). PTEN is a major negative regulator of the PI3K/Akt signaling pathway (1,4,5). PTEN possesses a carboxy-terminal, noncatalytic regulatory domain with three phosphorylation sites (Ser380, Thr382, and Thr383) that regulate PTEN stability and may affect its biological activity (6,7). PTEN regulates p53 protein levels and activity (8) and is involved in G protein-coupled signaling during chemotaxis (9,10).

The Phospho-Akt Pathway Antibody Sampler Kit provides an economical means to evaluate the activation status of the Akt signaling pathway, including PTEN and phosphorylated Akt, GSK-3beta, c-Raf and PDK1. The kit includes enough primary and secondary antibodies to perform two Western blot experiments.

Background: Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).

Phospho-Syk Sampler Kit provides an economical means to evaluate the activation status of Syk, including the phosphorylation of Tyr323, Tyr352 and Tyr525/526. The control Syk Antibody is also included. The kit contains enough primary and secondary antibodies for two Western blot experiments.

Background: Syk is a protein tyrosine kinase that plays an important role in intracellular signal transduction in hematopoietic cells (1-3). Syk interacts with immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic domains of immune receptors (4). It couples the activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation, differentiation, and phagocytosis (4). There is also evidence of a role for Syk in nonimmune cells and investigators have indicated that Syk is a potential tumor suppressor in human breast carcinomas (5). Tyr323 is a negative regulatory phosphorylation site within the SH2-kinase linker region in Syk. Phosphorylation at Tyr323 provides a direct binding site for the TKB domain of Cbl (6,7). Tyr352 of Syk is involved in the association of PLCγ1 (8). Tyr525 and Tyr526 are located in the activation loop of the Syk kinase domain; phosphorylation at Tyr525/526 of human Syk (equivalent to Tyr519/520 of mouse Syk) is essential for Syk function (9).

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 Nuclear Receptor Antibody Sampler Kit provides an economical means to evaluate the presence and status of nuclear receptors. This kit contains enough primary antibody to perform two western blots per primary.
The Innate Immunity Activation Antibody Sampler Kit provides an economical means of detecting the activation of multiple signaling pathways involved in innate immunity using phospho-specific, cleavage-specific, and control antibodies. The kit contains enough primary antibodies to perform at least two western blot experiments.

Background: The innate immune system responds rapidly to pathogens by detecting conserved pathogen-associated molecular patterns (PAMPs) and damage/danger-associated molecular patterns (DAMPs) through pattern recognition receptors (PRRs). There are several families of PRRs. Toll-like receptors (TLRs) are transmembrane PRRs and signal through recruitment of adaptor proteins, including MyD88, which leads to recruitment and phosphorylation of IRAK1 and IRAK4, followed by activation of NF-κB and MAP kinases (1-3). Some TLRs also activate IRFs, which upregulate the type I interferon response. Activation of TLR3 and TLR4 results in phosphorylation and activation of IRF-3, while TLR7, TLR8, and TLR9 lead to activation of IRF-7 (2, 3). STING is a multi-pass ER transmembrane protein that is activated in response to intracellular DNA downstream of DNA-sensing cytoplasmic PRRs, such as DDX41, or by binding the second messenger cyclic-GMP-AMP (cGAMP) produced by cGAS (4-6). Following activation, STING translocates with TBK1 to perinuclear endosomes, leading to phosphorylation and activation of IRF-3 and NF-κB (7, 8). Following activation and translocation, STING gets phosphorylated by ULK1, resulting in STING inactivation and degradation (9). Inflammasomes are cytoplasmic multimeric protein complexes that assemble in response to PAMPs or DAMPs detected by AIM2 or members of the nod-like receptor (NLR) family, such as NLRP3 (10). Inflammasomes activate Caspase-1, which cleaves the IL-1β and IL-18 precursor proteins into the mature forms (10).

The Huntingtin Interaction Antibody Sampler kit provides an economical means of detecting transcription-related proteins that interact with Huntingtin (Htt). This kit contains enough antibody to perform two western blot experiments per primary antibody.