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Product listing: HSP27 Antibody Sampler Kit, UniProt ID X10101 #12594 to Innate Immunity Activation Antibody Sampler Kit, UniProt ID P01584 #52239

The HSP27 Antibody Kit provides an economical means to evaluate the activation status of the HSP27 protein. The kit contains enough primary antibody to perform two western blot experiments per primary antibody.

Background: Heat shock protein (HSP) 27 is one of the small HSPs that are constitutively expressed at different levels in various cell types and tissues. Like other small HSPs, HSP27 is regulated at both the transcriptional and posttranslational levels (1). In response to stress, the HSP27 expression increases several-fold to confer cellular resistance to the adverse environmental change. HSP27 is phosphorylated at Ser15, Ser78, and Ser82 by MAPKAPK-2 as a result of the activation of the p38 MAP kinase pathway (2,3). Phosphorylation of HSP27 causes a change in its tertiary structure, which shifts from large homotypic multimers to dimers and monomers (4). It has been shown that phosphorylation and increased concentration of HSP27 modulates actin polymerization and reorganization (5,6).

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 Human T Cell Co-inhibitory and Co-stimulatory Receptor IHC Antibody Sampler Kit provides an economical means of detecting expression of receptors that modulate T cell activity in formalin-fixed, paraffin-embedded tissue samples.
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.
The IAP Family Antibody Sampler Kit provides an economical means to investigate the expression of various IAP family members within the cell. The kit contains enough primary and secondary antibodies to perform two Western blot experiments.

Background: The inhibitor of apoptosis protein (IAP) family consists of an evolutionarily conserved group of apoptosis inhibitors containing a conserved 70 amino acid BIR (baculovirus inhibitor repeat) domain (1,2). Human members of this family include c-IAP1, c-IAP2, XIAP, survivin, livin, and NAIP. Overexpression of IAP family members, particularly survivin and livin, in cancer cell lines and primary tumors suggests an important role for these proteins in cancer progression (3-5). In general, the IAP proteins function through direct interactions to inhibit the activity of several caspases, including caspase-3, caspase-7, and caspase-9 (5,6). In addition, binding of IAP family members to the mitochondrial protein Smac blocks their interaction with caspase-9, thereby allowing the processing and activation of the caspase (2).

$320
100 µg
This peptide is used to block IGF-I Receptor β Antibody #3027 reactivity in dot blot protocols.
APPLICATIONS

Application Methods: Immunohistochemistry (Paraffin)

Background: Insulin-like growth factor 1 (IGF-1) is a small (7.65 kDa) growth factor that interacts with both the IGF-1 receptor and the insulin receptor to control cell growth and apoptosis. Release of this endocrine hormone from the liver is stimulated by growth hormone produced in the anterior pituitary (1). Circulating IGF-1 is typically bound to one of six known IGF binding proteins (IGF-BP) (2). At target cells, the IGF-1 ligand binds IGF receptors (or insulin receptors) leading to receptor autophosphorylation and activation (3). Activated receptors mediate downstream signaling pathways (including Akt and MAPK) that regulate cell proliferation, apoptosis, development and longevity. Altered expression or mutation of IGF-1 is associated with several human disorders, including type I diabetes and various forms of cancer (4). Recombinant human IGF-1 has been used in clinical trials as a potential therapeutic agent in the treatment of human diseases (5).

Each control slide contains formalin fixed, paraffin-embedded Jurkat cells, both untreated and treated with etoposide, that serve as a control for cleaved caspase-3 (Asp 175) immunostaining. Western blot analysis was performed on extracts derived from the same cells to verify the efficacy of the etoposide treatment.To be used with antibodies: 9664, 9661, 9662, 2035, 9541.

Background: Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (1). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Cleavage of caspase-3 requires the aspartic acid residue at the P1 position (2).

Each control slide contains formalin fixed, paraffin-embedded HeLa cells, untreated, treated with Human Interferon-α1 (hIFN-α1) #8927 that serve as a control for Phospho-Stat1 (Tyr701) and Phospho-Stat3 (Tyr705) immunostaining. Western blot analysis was performed on extracts derived from the same cells to verify the efficacy of the hIFN-α1 treatment.
Each control slide contains formalin fixed, paraffin-embedded cell pellets, LNCaP (LKB1 positive) and A549 (LKB1 negative), that serve as a control for LKB1 immunostaining.

Background: LKB1 (STK11) is a serine/threonine kinase and tumor suppressor that helps control cell structure, apoptosis and energy homeostasis through regulation of numerous downstream kinases (1,2). A cytosolic protein complex comprised of LKB1, putative kinase STRAD, and the MO25 scaffold protein, activates both AMP-activated protein kinase (AMPK) and several AMPK-related kinases (3). AMPK plays a predominant role as the master regulator of cellular energy homeostasis, controlling downstream effectors that regulate cell growth and apoptosis in response to cellular ATP concentrations (4). LKB1 appears to be phosphorylated in cells at several sites, including human LKB1 at Ser31/325/428 and Thr189/336/363 (5).Mutation in the corresponding LKB1 gene causes Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder characterized by benign GI tract polyps and dark skin lesions of the mouth, hands, and feet (6). A variety of other LKB1 gene mutations have been associated with the formation of sporadic cancers in several tissues (7).

Each control slide contains formalin fixed, paraffin-embedded cell pellets: Raw 264.7 (mPD-L1 negative) and mouse bone marrow-derived macrophages (mPD-L1 positive), which serve as controls for mPD-L1 immunostaining.

Background: Programmed cell death 1 ligand 1 (PD-L1, B7-H1, CD274) is a member of the B7 family of cell surface ligands that regulate T cell activation and immune responses. The PD-L1 ligand binds the PD-1 transmembrane receptor and inhibits T cell activation. PD-L1 was discovered following a search for novel B7 protein homologs and was later shown to be expressed by antigen presenting cells, activated T cells, and tissues including placenta, heart, and lung (1-3). Similar in structure to related B7 family members, PD-L1 protein contains extracellular IgV and IgC domains and a short, cytoplasmic region. Research studies demonstrate that PD-L1 is expressed in several tumor types, including melanoma, ovary, colon, lung, breast, and renal cell carcinomas (4-6). Expression of PD-L1 in cancer is associated with tumor infiltrating lymphocytes, which mediate PD-L1 expression through the release of interferon gamma (7). Additional research links PD-L1 expression to cancers associated with viral infections (8,9).

Each control slide contains formalin fixed, paraffin-embedded HCT 116 cells, both untreated and treated with hTNF-α, that serve as a control for NF-κB p65 immunostaining.

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).

Each control slide contains formalin fixed, paraffin-embedded cell pellets, HDLM-2 (PD-L1 positive) and PC-3 (PD-L1 negative), that serve as controls for PD-L1 immunostaining.

Background: Programmed cell death 1 ligand 1 (PD-L1, B7-H1, CD274) is a member of the B7 family of cell surface ligands that regulate T cell activation and immune responses. The PD-L1 ligand binds the PD-1 transmembrane receptor and inhibits T cell activation. PD-L1 was discovered following a search for novel B7 protein homologs and was later shown to be expressed by antigen presenting cells, activated T cells, and tissues including placenta, heart, and lung (1-3). Similar in structure to related B7 family members, PD-L1 protein contains extracellular IgV and IgC domains and a short, cytoplasmic region. Research studies demonstrate that PD-L1 is expressed in several tumor types, including melanoma, ovary, colon, lung, breast, and renal cell carcinomas (4-6). Expression of PD-L1 in cancer is associated with tumor infiltrating lymphocytes, which mediate PD-L1 expression through the release of interferon gamma (7). Additional research links PD-L1 expression to cancers associated with viral infections (8,9).

Each control slide contains formalin fixed, paraffin-embedded LNCaP cells, both untreated and treated with PI3-Kinase inhibitor LY294002, that serve as a control for Phospho-Akt (Ser473) immunostaining. Western blot analysis was performed on extracts derived from the same cells to verify the efficacy of the LY942002 treatment.To be used with antibodies: 2855, 9644, 4060, 3787, 2938, 4691, 4685, 2920, 9323, 5482, 5196, 2997, 2691, 4858, 4857, 2211, 5364, 2217, 2317.

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).

Each control slide contains formalin fixed, paraffin-embedded KYSE 450 cells, both untreated and treated with EGF, that serve as a control for Phospho-EGFR immunostaining. Western blot analysis was performed on extracts derived from the same cells to verify the efficacy of the EGF treatment.To be used with antibodies: 2235, 2237, 3777, 2236, 2234, 4404, 4407, 4267, 9411, 9417, 9416.

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).

Each control slide contains formalin-fixed and paraffin-embedded SK-BR-3 cells, untreated and EGF-treated, that can serve as a control for immunostaining. Western blot analysis was performed on extracts derived from the same cells to verify treatment efficacy.To be used with antibodies: 3777,4407, 4267, 2243, 4290, 2165, 2242, 4791.
Each control slide contains formalin fixed, paraffin-embedded MKN45 cells, both untreated and treated with the c-Met inhibitor SU11274, that serve as a control for Phospho-Met (Tyr1234/1235) immunostaining. Western blot analysis was performed on extracts derived from the same cells to verify the efficacy of the SU11274 treatment.To be used with antibodies: 3077.

Background: Met, a high affinity tyrosine kinase receptor for hepatocyte growth factor (HGF, also known as scatter factor) is a disulfide-linked heterodimer made of 45 kDa α- and 145 kDa β-subunits (1,2). The α-subunit and the amino-terminal region of the β-subunit form the extracellular domain. The remainder of the β-chain spans the plasma membrane and contains a cytoplasmic region with tyrosine kinase activity. Interaction of Met with HGF results in autophosphorylation at multiple tyrosines, which recruit several downstream signaling components, including Gab1, c-Cbl, and PI3 kinase (3). These fundamental events are important for all of the biological functions involving Met kinase activity. The addition of a phosphate at cytoplasmic Tyr1003 is essential for Met protein ubiquitination and degradation (4). Phosphorylation at Tyr1234/1235 in the Met kinase domain is critical for kinase activation. Phosphorylation at Tyr1349 in the Met cytoplasmic domain provides a direct binding site for Gab1 (5). Research studies have shown that altered Met levels and/or tyrosine kinase activities are found in several types of tumors, including renal, colon, and breast. Thus, investigators have concluded that Met is an attractive potential cancer therapeutic and diagnostic target (6,7).

Each control slide contains formalin fixed, paraffin-embedded NIH/3T3 cells, treated with either U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) #9903 or TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 , that serve as a control for phospho-p44/42 MAPK (Thr202/Tyr204) immunostaining. U0126 has been shown to be a highly selective inhibitor of MEK1 and MEK2. TPA induces phosphorylation of p44/42 MAPK. Western blot analysis was performed on extracts derived from the same cells to verify the efficacy of the U0126 and TPA treatments.To be used with antibodies: 4370, 4376, 4695, 4696, 9102, 9108.

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.

Each control slide contains formalin fixed, paraffin-embedded LNCaP and NIH/3T3 cell pellets. NIH/3T3 cells express PTEN while LNCaP cells do not express PTEN. Western blot analysis was performed on extracts derived from the same cells to verify PTEN expression.To be used with antibodies: 9188, 9559.

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).

$448
50 sections
1 Kit
The PD-L1, CD3ε, CD8α Multiplex IHC Antibody Panel enables researchers to simultaneously detect these targets in paraffin-embedded tissues using tyramide signal amplification. Each antibody in the panel has been validated for this approach. For recommended staining conditions optimized specifically for this antibody panel please refer to Table 1 on the Data Sheet.
REACTIVITY
Human

Background: The field of cancer immunotherapy is focused on empowering the immune system to fight cancer. This approach has seen recent success in the clinic with targeting immune checkpoint control proteins, such as PD-1 (1,2). Despite this success, clinical biomarkers that predict response to therapeutic strategies involving PD-1 receptor blockade are still under investigation (3-5). While PD-L1 expression has been linked with an increased likelihood of response to anti-PD-1 therapy, research studies have shown that additional factors, such as tumor-immune infiltration and the ratio of effector to regulatory T cells within the tumor, could play a significant role in predicting treatment outcome (6-9).

$448
50 sections
1 Kit
The PD-L1, FoxP3, CD8α Multiplex IHC Antibody Panel enables researchers to simultaneously detect these targets in paraffin-embedded tissues using tyramide signal amplification. Each antibody in the panel has been validated for this approach. For recommended staining conditions optimized specifically for this antibody panel please refer to Table 1 on the Data Sheet.
REACTIVITY
Human

Background: The field of cancer immunotherapy is focused on empowering the immune system to fight cancer. This approach has seen recent success in the clinic with targeting immune checkpoint control proteins, such as PD-1 (1,2). Despite this success, clinical biomarkers that predict response to therapeutic strategies involving PD-1 receptor blockade are still under investigation (3-5). While PD-L1 expression has been linked with an increased likelihood of response to anti-PD-1 therapy, research studies have shown that additional factors, such as tumor-immune infiltration and the ratio of effector to regulatory T cells within the tumor, could play a significant role in predicting treatment outcome (6-9).

$499
4 x 40 µl
1 Kit
The SignalStain® Akt Pathway IHC Sampler Kit from Cell Signaling Technology allows the researcher to examine paraffin-embedded tissues or cells with antibodies directed against proteins involved in Akt signaling. Multiple approaches are used to validate each antibody for use in immunohistochemical assays. Also included in the kit are control slides that can be used to verify the performance of each antibody and a primary antibody diluent. Please see the above table for the recommended antibody diluent for each kit antibody.
APPLICATIONS

Application Methods: Immunohistochemistry (Paraffin)

$499
120 slides
1 Kit
SignalStain® Apoptosis (Cleaved Caspase-3) IHC Detection Kit allows the detection of activated caspase-3 in formalin-fixed paraffin-embedded human and mouse tissue samples. Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb is detected by the polymer based, HRP-conjugated SignalStain® Boost IHC Detection Reagent in combination with SignalStain® DAB Diluent and Chromogen Concentrate. Also included is a concentration-matched rabbit monoclonal IgG control to verify the specificity of staining.This combination of reagents provides a sensitive and specific means of detecting apoptotic events in tissue samples.
APPLICATIONS
REACTIVITY
Human, Mouse

Application Methods: Immunohistochemistry (Paraffin)

Background: Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (1). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Cleavage of caspase-3 requires the aspartic acid residue at the P1 position (2).

$499
4 x 40 µl
1 Kit
The SignalStain® Phospho-ErbB Family IHC Sampler Kit from Cell Signaling Technology allows the researcher to examine paraffin-embedded tissues or cells with antibodies that will detect active ErbB 1, 2 and 3 as well as total epidermal growth factor receptor (EGFR). Each antibody is validated for use in immunohistochemical assays using multiple approaches. Also included in the kit are control slides that can be used to verify the performance of each antibody and a primary antibody diluent. See the table above for the recommended antibody diluent for each antibody provided in the kit.
APPLICATIONS

Application Methods: Immunohistochemistry (Paraffin)

$499
4 x 40 µl
1 Kit
The SignalStain® Proliferation/Apoptosis IHC Sampler Kit from Cell Signaling Technology allows the researcher to examine paraffin-embedded tissues or cells with antibodies that will detect cellular apoptosis or proliferation. Each antibody is validated for use in immunohistochemical assays using multiple approaches. Also included in the kit are control slides that can be used to verify the performance of each antibody and a primary antibody diluent. Please see table above for recommended diluent for each antibody in this kit.
APPLICATIONS

Application Methods: Immunohistochemistry (Paraffin)

The IKK Isoform Antibody Sampler Kit provides an economical means to investigate NFkappaB signaling within the cell. The kit contains primary and secondary antibodies to perform two Western blots with each antibody.

Background: The NF-κB/Rel transcription factors are present in the cytosol in an inactive state, complexed with the inhibitory IκB proteins (1-3). Most agents that activate NF-κB do so through a common pathway based on phosphorylation-induced, proteasome-mediated degradation of IκB (3-7). The key regulatory step in this pathway involves activation of a high molecular weight IκB kinase (IKK) complex whose catalysis is generally carried out by three tightly associated IKK subunits. IKKα and IKKβ serve as the catalytic subunits of the kinase and IKKγ serves as the regulatory subunit (8,9). Activation of IKK depends upon phosphorylation at Ser177 and Ser181 in the activation loop of IKKβ (Ser176 and Ser180 in IKKα), which causes conformational changes, resulting in kinase activation (10-13).

$91
100 ml
This product is supplied as a 1X working solution for antibody dilution in immunofluorescence assays with cell cultures (IF-IC) or frozen tissue samples (IF-F). Cell Signaling Technology recommends using this buffer according to our protocols for IF-approved primary antibodies to ensure accurate and reproducible results. This product contains enough material for 500 assays based on a 100 μl assay volume.
APPLICATIONS

Application Methods: Immunofluorescence (Frozen), Immunofluorescence (Immunocytochemistry)

$39
50 ml
This blocking reagent is designed to reduce noise originating from nonspecific protein-protein interactions in immunofluorescence assays. The core formulation includes goat serum as a protein blocker mixed with a mild detergent to facilitate permeabilization of cellular membranes.Cell Signaling Technology recommends using the buffer in accordance with our protocols for cultured cells (IF-IC) and frozen tissue sections (IF-F) to ensure accurate and reproducible results.The product is supplied as a 1X working solution and contains enough material for 500 assays based on a 100 μl assay volume.
APPLICATIONS

Application Methods: Immunofluorescence (Frozen), Immunofluorescence (Immunocytochemistry)

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.

The Initiator Caspases Antibody Sampler Kit provides an economical means of evaluating initiator (apical) caspase proteins. The kit contains enough primary antibody to perform two western blots with each primary antibody.
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).