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Antibody Sampler Kit Regulation of Mapkkk Cascade

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 Receptor Tyrosine Kinase Antibody Sampler Kit provides the means to detect a broad range of common receptor tyrosine kinases, as well as total phospho-tyrosine activity. The kit provides enough antibody to perform two western blot experiments with each primary antibody.
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 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-EGF Receptor Pathway Sampler Kit provides an economical means to evaluate the activation status of multiple members of the EGF receptor pathway, including phosphorylated EGF receptor, Stat5, c-Cbl, Shc, Gab1, PLCγ1, Akt and p44/42 MAPK. The kit includes enough primary and secondary antibodies to perform two western blot experiments.

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).

The Phospho-MAPK Family Antibody Sampler Kit provides an economical means of evaluating the phosphorylation state of p38, p44/42, and SAPK/JNK mitogen-activated protein kinases. The kit contains enough primary and secondary antibodies to perform two western blot experiments.

Background: p44/42 MAPK (Erk1/2), SAPK/JNK, and p38 MAPK function in protein kinase cascades that play a critical role in the regulation of cell growth, differentiation, and control of cellular responses to cytokines and stress. p44/42 MAPK is activated by growth and neurotrophic factors. Activation occurs through phosphorylation of threonine and tyrosine residues (Thr202 and Tyr204 in human Erk1) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK). SAPK/JNK and p38 MAPK are activated by inflammatory cytokines and by a wide variety of cellular stresses. Activation of SAPK/JNK occurs via phosphorylation at Thr183 and Tyr185 by the dual specificity enzyme SEK/MKK4. Both MKK3 and SEK phosphorylate p38 MAPK on tyrosine and threonine at the sequence T*GY* to activate p38 MAP kinase (1-5).

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).

This sampler kit provides an economical means to investigate protein folding and stability. The kit contains primary and secondary antibodies to perform two Western blots with each antibody.
The MAPK Family Antibody Sampler Kit provides an economical means of evaluating total levels of p38, p44/42, and SAPK/JNK mitogen-activated protein kinases. The kit contains enough primary and secondary antibody to perform two western blot experiments.

Background: p44/42 MAPK (Erk1/2), SAPK/JNK, and p38 MAPK function in protein kinase cascades that play a critical role in the regulation of cell growth, differentiation, and control of cellular responses to cytokines and stress. p44/42 MAPK is activated by growth and neurotrophic factors. Activation occurs through phosphorylation of threonine and tyrosine residues (Thr202 and Tyr204 in human Erk1) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK). SAPK/JNK and p38 MAPK are activated by inflammatory cytokines and by a wide variety of cellular stresses. Activation of SAPK/JNK occurs via phosphorylation at Thr183 and Tyr185 by the dual specificity enzyme SEK/MKK4. Both MKK3 and SEK phosphorylate p38 MAPK on tyrosine and threonine at the sequence T*GY* to activate p38 MAP kinase (1-5).

Background: Autophagy is a catabolic process for the autophagosome-lysosomal degradation of bulk cytoplasmic contents (1, 2). Selective autophagy targets the degradation of distinct sets of substrates and organelles (3-5). One of the best studied examples of selective autophagy involves the clearance of damaged mitochondria through a process called mitophagy. Several pathways have been described for various contexts of mitophagy, including the FUNDC1 pathway, the BNIP3 and BNIP3L/Nix pathway, and the PINK1/Parkin pathway. FUNDC1 is a mitochondrial protein that is phosphorylated by the autophagy kinase ULK1 and regulates hypoxia induced mitophagy (6, 7). BNIP3L/Nix and BNIP3 are members of the Bcl-2 family of apoptosis regulators that are expressed on mitochondria, induced by hypoxia, and have have been shown to play a role in mitophagy (8). BNIP3L/Nix is also important in the autophagic maturation of erythroid cells (9). FUNDC1, BNIP3 and BNIP3L/Nix bind to LC3 family members, targeting the mitochondria to the autophagosome.Non-hypoxic induction of mitophagy can be regulated by the PINK1/Parkin pathway, which plays causative roles in neurodegenerative disease, most notably Parkinson’s disease (10, 11). PINK1 is a mitochondrial serine/threonine kinase that is stabilized on the outer mitochondrial membrane of damaged mitochondria. Substrates of PINK1 include the E3 ubiquitin ligase Parkin and ubiquitin itself (12-14). Phosphorylation of Parkin as well as binding to phosphorylated ubiquitin leads to accumulation of ubiquitinated chains on multiple mitochondrial proteins. Ubiquitinated proteins are recognized by selective cargo receptors including SQSTM1/p62, Optineurin, and NDP52 (15-16). Autophagy cargo receptors contain an LC3-interacting region (LIR) required for binding to Atg8/LC3 family members and targeting to the autophagosome (3).

The PDGF Receptor Activation Antibody Sampler Kit provides an economical means to evaluate the activation status of multiple members of the PDGF receptor pathway, including SHP-2, Akt, and p44/42 MAPK (Erk1/2). The kit includes enough antibody to perform two western blot experiments per primary antibody.
The Raf Family Antibody Sampler Kit provides a fast and economical means to investigate Raf signaling. The kit contains enough primary and secondary antibody to perform two Western blot experiments.

Background: A-Raf, B-Raf, and c-Raf (Raf-1) are the main effectors recruited by GTP-bound Ras to activate the MEK-MAP kinase pathway (1). Activation of c-Raf is the best understood and involves phosphorylation at multiple activating sites including Ser338, Tyr341, Thr491, Ser494, Ser497, and Ser499 (2). p21-activated protein kinase (PAK) has been shown to phosphorylate c-Raf at Ser338, and the Src family phosphorylates Tyr341 to induce c-Raf activity (3,4). Ser338 of c-Raf corresponds to similar sites in A-Raf (Ser299) and B-Raf (Ser445), although this site is constitutively phosphorylated in B-Raf (5). Inhibitory 14-3-3 binding sites on c-Raf (Ser259 and Ser621) can be phosphorylated by Akt and AMPK, respectively (6,7). While A-Raf, B-Raf, and c-Raf are similar in sequence and function, differential regulation has been observed (8). Of particular interest, B-Raf contains three consensus Akt phosphorylation sites (Ser364, Ser428, and Thr439) and lacks a site equivalent to Tyr341 of c-Raf (8,9). Research studies have shown that the B-Raf mutation V600E results in elevated kinase activity and is commonly found in malignant melanoma (10). Six residues of c-Raf (Ser29, Ser43, Ser289, Ser296, Ser301, and Ser642) become hyperphosphorylated in a manner consistent with c-Raf inactivation. The hyperphosphorylation of these six sites is dependent on downstream MEK signaling and renders c-Raf unresponsive to subsequent activation events (11).

The Stress and Apoptosis Antibody Sampler Kit provides an economical means of evaluating stress and apoptotic responses of each protein. The kit contains enough primary and secondary antibody to perform two western blot experiments per primary antibody.
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 Mutant Ras Antibody Sampler Kit provides an economical means of detecting common mutation in the Ras protein. The kit contains enough primary and secondary antibodies to perform at least two western blot experiments.

Background: The 21 kDa guanine-nucleotide binding proteins (K-Ras, H-Ras, and N-Ras) cycle between active (GTP-bound) and inactive (GDP-bound) forms (1). Receptor tyrosine kinases and G protein-coupled receptors activate Ras, which then stimulates the Raf-MEK-MAPK pathway (2-4). GTPase-activating proteins (GAP) normally facilitate the inactivation of Ras. However, research studies have shown that in 30% of human tumors, point mutations in Ras prevent the GAP-mediated inhibition of this pathway (5). The most common oncogenic Ras mutation found in tumors is Gly12 to Asp12 (G12D), which prevents Ras inactivation, possibly by increasing the overall rigidity of the protein (5,6). Additional oncogenic mutations of Ras have been observed at varying frequencies at codons 12, 13, and 61. The Gly12 to Val12 (G12V) mutation has been detected in a number of different cancers, including colorectal and thyroid cancer (7,8).

The PDGF Receptor α Antibody Sampler Kit provides an economical means of evaluating total PDGF receptor α protein (PDGFRα) levels as well as PDGFRα phosphorylated at specific sites. The kit contains enough primary and secondary antibody to perform two western blots with each antibody.

Background: Platelet derived growth factor (PDGF) family proteins exist as several disulphide-bonded, dimeric isoforms (PDGF AA, PDGF AB, PDGF BB, PDGF CC, and PDGF DD) that bind in a specific pattern to two closely related receptor tyrosine kinases, PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ). PDGFRα and PDGFRβ share 75% to 85% sequence homology between their two intracellular kinase domains, while the kinase insert and carboxy-terminal tail regions display a lower level (27% to 28%) of homology (1). PDGFRα homodimers bind all PDGF isoforms except those containing PDGF D. PDGFRβ homodimers bind PDGF BB and DD isoforms, as well as the PDGF AB heterodimer. The heteromeric PDGF receptor α/β binds PDGF B, C, and D homodimers, as well as the PDGF AB heterodimer (2). PDGFRα and PDGFRβ can each form heterodimers with EGFR, which is also activated by PDGF (3). Various cells differ in the total number of receptors present and in the receptor subunit composition, which may account for responsive differences among cell types to PDGF binding (4). Ligand binding induces receptor dimerization and autophosphorylation, followed by binding and activation of cytoplasmic SH2 domain-containing signal transduction molecules, such as GRB2, Src, GAP, PI3 kinase, PLCγ, and NCK. A number of different signaling pathways are initiated by activated PDGF receptors and lead to control of cell growth, actin reorganization, migration, and differentiation (5). Tyr751 in the kinase-insert region of PDGFRβ is the docking site for PI3 kinase (6). Phosphorylated pentapeptides derived from Tyr751 of PDGFRβ (pTyr751-Val-Pro-Met-Leu) inhibit the association of the carboxy-terminal SH2 domain of the p85 subunit of PI3 kinase with PDGFRβ (7). Tyr740 is also required for PDGFRβ-mediated PI3 kinase activation (8).

The p38 MAPK Isoform Activation Antibody Sampler Kit provides an economical means to evaluate the activation status of individual isoforms of p38 MAPK through immunoprecipitation of the phosphorylated p38 MAPK followed by western blot using isoform specific antibodies. The kit includes enough primary and secondary antibodies to perform two IP/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).

The Cell Fractionation Antibody Sampler Kit provides an economical means for determining the purity of each distinctly separated subcellular fraction by western blot using Cell Signaling Technology's Cell Fractionation Kit #9038. This antibody sampler kit includes enough primary antibody to perform at least two western blots per primary antibody.
Branched Ubiquitin Antibody Sampler Kit provides an economical means of detecting total and common branch specific forms of ubiquitin. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Background: Ubiquitin is a conserved polypeptide unit that plays an important role in the ubiquitin-proteasome pathway. Ubiquitin can be covalently linked to many cellular proteins by the ubiquitination process, which targets proteins for degradation by the 26S proteasome. Three components are involved in the target protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (1-3). The ubiquitin-proteasome pathway has been implicated in a wide range of normal biological processes and in disease-related abnormalities. Several proteins such as IκB, p53, cdc25A, and Bcl-2 have been shown to be targets for the ubiquitin-proteasome process as part of regulation of cell cycle progression, differentiation, cell stress response, and apoptosis (4-7).

The Silent Synapses Antibody Sampler Kit provides an economical means of detecting the activation of AMPA-type glutamate receptors (AMPAR) using phospho-specific and control antibodies. AMPARs expression can be compared to other synaptic components including NMDA-type glutamate receptor subunit GluN1 and the synaptic scaffolding protein PSD95. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

Background: AMPA- (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), kainate-, and NMDA- (N-methyl-D-aspartate) receptors are the three main families of ionotropic glutamate-gated ion channels. AMPA receptors (AMPARs) are composed of four subunits (GluA1-4), which assemble as homo- or hetero-tetramers to mediate the majority of fast excitatory transmissions in the central nervous system. AMPARs are implicated in synapse formation, stabilization, and plasticity (1). In contrast to GluA2-containing AMPARs, AMPARs that lack GluA2 are permeable to calcium (2). Post-transcriptional modifications (alternative splicing, nuclear RNA editing) and post-translational modifications (glycosylation, phosphorylation) result in a very large number of permutations, fine-tuning the kinetic properties and surface expression of AMPARs representing key pathways to mediate synaptic plasticity (3). During development and mature states, some synapses exhibit “silent synapses” that lack functional AMPAR-mediated transmission. Synapses become “unsilenced” by post-translational modification of GluAs, particularly GluA1, which alters its kinetic properties and/or surface expression while other synaptic components, such as other glutamate receptors like NMDARs and postsynaptic scaffolding proteins like PSD95, remain unaltered. Conversely, reducing the AMPAR kinetic properties and surface expression can silence synapses. Key post-translational modifications implicated in regulating these processes include phosphorylation of GluA1 at Ser831 and Ser845 (4). Research studies have implicated activity-dependent changes in AMPARs in a variety of diseases, including Alzheimer’s, amyotrophic lateral sclerosis (ALS), stroke, and epilepsy (1).