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Product listing: SignalSilence® HER2/ErbB2 siRNA I, UniProt ID P04626 #6282 to Thapsigargin #12758

$262
3 nmol
300 µl
SignalSilence® HER2/ErbB2 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit HER2/ErbB2 expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
REACTIVITY
Human

Background: The ErbB2 (HER2) proto-oncogene encodes a 185 kDa transmembrane, receptor-like glycoprotein with intrinsic tyrosine kinase activity (1). While ErbB2 lacks an identified ligand, ErbB2 kinase activity can be activated in the absence of a ligand when overexpressed and through heteromeric associations with other ErbB family members (2). Amplification of the ErbB2 gene and overexpression of its product are detected in almost 40% of human breast cancers (3). Binding of the c-Cbl ubiquitin ligase to ErbB2 at Tyr1112 leads to ErbB2 poly-ubiquitination and enhances degradation of this kinase (4). ErbB2 is a key therapeutic target in the treatment of breast cancer and other carcinomas and targeting the regulation of ErbB2 degradation by the c-Cbl-regulated proteolytic pathway is one potential therapeutic strategy. Phosphorylation of the kinase domain residue Tyr877 of ErbB2 (homologous to Tyr416 of pp60c-Src) may be involved in regulating ErbB2 biological activity. The major autophosphorylation sites in ErbB2 are Tyr1248 and Tyr1221/1222; phosphorylation of these sites couples ErbB2 to the Ras-Raf-MAP kinase signal transduction pathway (1,5).

$262
3 nmol
300 µl
SignalSilence® HER2/ErbB2 siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit HER2/ErbB2 expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
REACTIVITY
Human

Background: The ErbB2 (HER2) proto-oncogene encodes a 185 kDa transmembrane, receptor-like glycoprotein with intrinsic tyrosine kinase activity (1). While ErbB2 lacks an identified ligand, ErbB2 kinase activity can be activated in the absence of a ligand when overexpressed and through heteromeric associations with other ErbB family members (2). Amplification of the ErbB2 gene and overexpression of its product are detected in almost 40% of human breast cancers (3). Binding of the c-Cbl ubiquitin ligase to ErbB2 at Tyr1112 leads to ErbB2 poly-ubiquitination and enhances degradation of this kinase (4). ErbB2 is a key therapeutic target in the treatment of breast cancer and other carcinomas and targeting the regulation of ErbB2 degradation by the c-Cbl-regulated proteolytic pathway is one potential therapeutic strategy. Phosphorylation of the kinase domain residue Tyr877 of ErbB2 (homologous to Tyr416 of pp60c-Src) may be involved in regulating ErbB2 biological activity. The major autophosphorylation sites in ErbB2 are Tyr1248 and Tyr1221/1222; phosphorylation of these sites couples ErbB2 to the Ras-Raf-MAP kinase signal transduction pathway (1,5).

$262
3 nmol
300 µl
SignalSilence® HER3/ErbB3 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit HER3/ErbB3 expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
REACTIVITY
Human

Background: HER3/ErbB3 is a member of the ErbB receptor protein tyrosine kinase family, but it lacks tyrosine kinase activity. Tyrosine phosphorylation of ErbB3 depends on its association with other ErbB tyrosine kinases. Upon ligand binding, heterodimers form between ErbB3 and other ErbB proteins, and ErbB3 is phosphorylated on tyrosine residues by the activated ErbB kinase (1,2). There are at least 9 potential tyrosine phosphorylation sites in the carboxy-terminal tail of ErbB3. These sites serve as consensus binding sites for signal transducing proteins, including Src family members, Grb2, and the p85 subunit of PI3 kinase, which mediate ErbB downstream signaling (3). Both Tyr1222 and Tyr1289 of ErbB3 reside within a YXXM motif and participate in signaling to PI3K (4).Investigators have found that ErbB3 is highly expressed in many cancer cells (5) and activation of the ErbB3/PI3K pathway is correlated with malignant phenotypes of adenocarcinomas (6). Research studies have demonstrated that in tumor development, ErbB3 may function as an oncogenic unit together with other ErbB members (e.g. ErbB2 requires ErbB3 to drive breast tumor cell proliferation) (7). Thus, investigators view inhibiting interaction between ErbB3 and ErbB tyrosine kinases as a novel strategy for anti-tumor therapy.

$262
3 nmol
300 µl
SignalSilence® HER3/ErbB3 siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit HER3/ErbB3 expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
REACTIVITY
Human

Background: HER3/ErbB3 is a member of the ErbB receptor protein tyrosine kinase family, but it lacks tyrosine kinase activity. Tyrosine phosphorylation of ErbB3 depends on its association with other ErbB tyrosine kinases. Upon ligand binding, heterodimers form between ErbB3 and other ErbB proteins, and ErbB3 is phosphorylated on tyrosine residues by the activated ErbB kinase (1,2). There are at least 9 potential tyrosine phosphorylation sites in the carboxy-terminal tail of ErbB3. These sites serve as consensus binding sites for signal transducing proteins, including Src family members, Grb2, and the p85 subunit of PI3 kinase, which mediate ErbB downstream signaling (3). Both Tyr1222 and Tyr1289 of ErbB3 reside within a YXXM motif and participate in signaling to PI3K (4).Investigators have found that ErbB3 is highly expressed in many cancer cells (5) and activation of the ErbB3/PI3K pathway is correlated with malignant phenotypes of adenocarcinomas (6). Research studies have demonstrated that in tumor development, ErbB3 may function as an oncogenic unit together with other ErbB members (e.g. ErbB2 requires ErbB3 to drive breast tumor cell proliferation) (7). Thus, investigators view inhibiting interaction between ErbB3 and ErbB tyrosine kinases as a novel strategy for anti-tumor therapy.

$262
3 nmol
300 µl
SignalSilence® Met siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Met expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
REACTIVITY
Human

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).

$262
3 nmol
300 µl
SignalSilence® Met siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit Met expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.
REACTIVITY
Human

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).

The Griess Reagent Nitrite Measurement Kit can be used to indirectly detect nitric oxide (NO) through the measurement one of its stable oxidation products, nitrite. Nitrite reacts with sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride (NED) to yield a pink azo dye. The azo dye produced in this assay can be measured spectrophotometrically using its absorbance at 550 nM.
The Malachite Green Phosphate Detection Kit is a convenient and sensitive, single-step free-phosphate determination kit that can be used for measuring phosphate released during enzymatic phosphatase assays.
The Resazurin Cell Viability Kit is a fluorescent assay that detects cellular metabolic activity. The blue nonfluorescent resazurin reagent is reduced to highly fluorescent resorufin by dehydrogenase enzymes in metabolically active cells. This conversion only occurs in viable cells and thus, the amount of resorufin produced is proportional to the number of viable cells in the sample. The resorufin formed in the assay can be quantified by measuring the relative fluorescence units (RFU) using a fluorometer (Ex=530-570 nm, Em=590-620 nm).
The PTMScan® Lys-C Digested Control Peptides I are produced from mouse liver tissue that has been lysed, reduced and alkylated, digested with Lys-C, purified, and lyophilized. This is intended to be used as a control for PTMScan® kits. It should only be used for phosphorylated motifs that contain lysine or arginine residues within the context of the antibody motif.
The PTMScan® Trypsin Digested Control Peptides I are produced from mouse liver tissue that has been lysed, reduced and alkylated, digested with trypsin, purified, and lyophilized. This is intended to be used as a control for PTMScan® kits. It should not be used for phosphorylated motifs that contain lysine or arginine residues within the context of the antibody motif.
$325
200 assays, 96 well format
1 Kit
The Cellular Glutathione Detection Assay Kit employs the cell permeable dye monochlorobimane (MCB) to detect reduced glutathione (GSH) in cellular assays. MCB displays a high affinity for reduced glutathione and exhibits a very low fluorescent yield when free in solution. Upon binding to GSH, the dye exhibits a strong blue fluorescence that can be measured at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Fluorescent intensity correlates with sample GSH level. This kit can be used to either label cells directly or to detect GSH level in cell extracts. The assay can be easily applied in high throughput plate-format, flow cytometry, or fluorescent imaging.
APPLICATIONS
REACTIVITY
All Species Expected

Application Methods: Flow Cytometry

PTMScan® Phospho-Enrichment IMAC Fe-NTA Magnetic Beads employ immobilized metal affinity chromatography for capturing phosphorylated peptides. Negatively charged phosphate groups are attracted to the positively charged metal ions on the beads. In conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS), this approach enables researchers to isolate, identify, and quantitate large numbers of phosphorylated cellular peptides with a high degree of specificity and sensitivity, providing a global overview of phosphorylation in cell and tissue samples. For more information on PTMScan® Proteomics Services, please visit https://www.cellsignal.com/services/index.html.
$271
500 assays (96 well format)
1 Kit
The Mitochondrial Membrane Potential Assay Kit (II) is a fluorescent assay that detects the mitochondrial membrane potential in living cells. The kit includes the cationic dye TMRE (tetramethylrhodamine ethyl ester perchlorate) and a mitochondrial membrane potential disruptor CCCP (carbonyl cyanide 3-chlorophenylhydrazone). TMRE is a cell membrane permeable, fluorescent dye that accumulates in intact mitochondria. Depolarized or inactive mitochondria exhibit decreased membrane potential, resulting in reduced TMRE accumulation.
APPLICATIONS

Application Methods: Flow Cytometry, Immunofluorescence (Immunocytochemistry)

$303
100 µl
APPLICATIONS
REACTIVITY
All Species Expected

Application Methods: Western Blotting

$489
96 assays
1 Kit
PathScan® Phospho-c-Abl (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated Bcr-Abl and c-Abl proteins. A c-Abl rabbit antibody has been coated on the microwells. After incubation with cell lysates, Bcr-Abl and c-Abl protein (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, a phospho-tyrosine mouse detection antibody is added to detect captured tyrosine-phosphorylated Bcr-Abl and c-Abl protein. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of tyrosine-phosphorylated Bcr-Abl and c-Abl protein.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human

Background: The c-Abl proto-oncogene encodes a nonreceptor protein tyrosine kinase that is ubiquitously expressed and highly conserved in metazoan evolution. c-Abl protein is distributed in both the nucleus and the cytoplasm of cells. It is implicated in regulating cell proliferation, differentiation, apoptosis, cell adhesion, and stress responses (1-3). c-Abl kinase activity is increased in vivo by diverse physiological stimuli including integrin activation; PDGF stimulation; and binding to c-Jun, Nck, and RFX1 (2,4). The in vivo mechanism for regulation of c-Abl kinase activity is not completely understood. Tyr245 is located in the linker region between the SH2 and catalytic domains. This positioning is conserved among Abl family members. Phosphorylation at Tyr245 is involved in the activation of c-Abl kinase (5). In addition, phosphorylation at Tyr412, which is located in the kinase activation loop of c-Abl, is required for kinase activity (6).

$489
96 assays
1 Kit
The PathScan® Phospho-EGF Receptor (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated EGF receptor protein. An EGF Receptor Rabbit mAb has been coated on the microwells. After incubation with cell lysates, EGF receptor protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Phospho-Tyrosine Mouse Detection mAb is added to detect captured tyrosine-phosphorylated EGF receptor protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of EGF receptor protein phosphorylated at tyrosine residues.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human

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).

$489
96 assays
1 Kit
The PathScan® Phospho-GSK-3β (Ser9) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of GSK-3β protein phosphorylated at Ser9. A GSK-3β mouse mAb has been coated onto the microwells. After incubation with cell lysates, GSK-3β (phospho and nonphospho) protein is captured by the coated antibody. Following extensive washing, a phospho-GSK-3β (Ser9) rabbit mAb is added to detect the captured phospho-GSK-3β protein. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of GSK-3β phosphorylated at Ser9.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Monkey, Mouse, Rat

Background: Glycogen synthase kinase-3 (GSK-3) was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (1). GSK-3 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates and inactivates glycogen synthase. GSK-3 is a critical downstream element of the PI3K/Akt cell survival pathway whose activity can be inhibited by Akt-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β (2,3). GSK-3 has been implicated in the regulation of cell fate in Dictyostelium and is a component of the Wnt signaling pathway required for Drosophila, Xenopus, and mammalian development (4). GSK-3 has been shown to regulate cyclin D1 proteolysis and subcellular localization (5).

$489
96 assays
1 Kit
The PathScan® Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that recognizes endogenous levels of phospho-Smad2 (Ser465/467) and Smad3 (Ser423/425) proteins. A Smad2/3 Mouse Antibody has been coated on the microwells. After incubation with cell lysates, Smad2/3 proteins (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, a Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) Detection Antibody is added to detect captured phospho-Smad2 (Ser465/467) and phospho-Smad3 (Ser423/425) proteins. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of phospho-Smad2 (Ser465/467) and phospho-Smad3 (Ser423/425) proteins.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mink, Mouse

Background: Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, and 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).

$489
96 assays
1 Kit
The PathScan® Phospho-Smad3 (Ser423/425) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that recognizes endogenous levels of Smad3 (Ser423/425) protein. A Smad2/3 Mouse Antibody has been coated on the microwells. After incubation with cell lysates, Smad3 proteins (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, a Phospho-Smad3 (Ser423/425) Rabbit Detection Antibody is added to detect captured phospho-Smad3 (Ser423/425) proteins. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of phospho-Smad3 (Ser423/425) proteins.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mink, Mouse

Background: Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, and 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).

$489
96 assays
1 Kit
The PathScan® Total Cyclin D1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total cyclin D1 protein. A Cyclin D1 Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, both phospho and nonphospho cyclin D1 proteins are captured by the coated antibody. Following extensive washing, Cyclin D1 Mouse Detection Antibody is added to detect the captured cyclin D1 protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of total cyclin D1 protein.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Monkey, Mouse, Rat

Background: Activity of the cyclin-dependent kinases CDK4 and CDK6 is regulated by T-loop phosphorylation, by the abundance of their cyclin partners (the D-type cyclins), and by association with CDK inhibitors of the Cip/Kip or INK family of proteins (1). The inactive ternary complex of cyclin D/CDK4 and p27 Kip1 requires extracellular mitogenic stimuli for the release and degradation of p27 concomitant with a rise in cyclin D levels to affect progression through the restriction point and Rb-dependent entry into S-phase (2). The active complex of cyclin D/CDK4 targets the retinoblastoma protein for phosphorylation, allowing the release of E2F transcription factors that activate G1/S-phase gene expression (3). Levels of cyclin D protein drop upon withdrawal of growth factors through downregulation of protein expression and phosphorylation-dependent degradation (4).

$489
96 assays
1 Kit
The PathScan® Total GSK-3β Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of GSK-3β protein. A GSK-3β mouse mAb has been coated onto the microwells. After incubation with cell lysates, GSK-3β (phospho and nonphospho) protein is captured by the coated antibody. Following extensive washing, a GSK-3β rabbit mAb is added to detect the captured GSK-3β protein. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of total GSK-3β.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Monkey, Mouse, Rat

Background: Glycogen synthase kinase-3 (GSK-3) was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (1). GSK-3 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates and inactivates glycogen synthase. GSK-3 is a critical downstream element of the PI3K/Akt cell survival pathway whose activity can be inhibited by Akt-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β (2,3). GSK-3 has been implicated in the regulation of cell fate in Dictyostelium and is a component of the Wnt signaling pathway required for Drosophila, Xenopus, and mammalian development (4). GSK-3 has been shown to regulate cyclin D1 proteolysis and subcellular localization (5).

$489
96 assays
1 Kit
The PathScan® Total Oct-4A Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Oct-4A. An Oct-4A Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, Oct-4A protein is captured by the coated antibody. Following extensive washing, an Oct-4A Mouse Detection Antibody is added to detect the captured Oct-4A protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of Oct-4A.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human

Background: Oct-4 (POU5F1) is a transcription factor highly expressed in undifferentiated embryonic stem cells and embryonic germ cells (1). A network of key factors that includes Oct-4, Nanog, and Sox2 is necessary for the maintenance of pluripotent potential, and downregulation of Oct-4 has been shown to trigger cell differentiation (2,3). Research studies have demonstrated that Oct-4 is a useful germ cell tumor marker (4). Oct-4 exists as two splice variants, Oct-4A and Oct-4B (5). Recent studies have suggested that the Oct-4A isoform has the ability to confer and sustain pluripotency, while Oct-4B may exist in some somatic, non-pluripotent cells (6,7).

$489
96 assays
1 Kit
The PathScan® Total Smad2/3 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that recognizes endogenous levels of Smad2 and Smad3 proteins. A Smad2/3 Mouse Antibody has been coated on the microwells. After incubation with cell lysates, Smad2/3 proteins (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, a Smad2/3 Rabbit Detection Antibody is added to detect captured Smad2/3 proteins. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Smad2 and Smad3 proteins.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mink, Mouse

Background: Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, and 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).

$489
96 assays
1 Kit
The PathScan® Total Smad3 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that recognizes endogenous levels of Smad3 protein. A Smad3 Rabbit Antibody has been coated on the microwells. After incubation with cell lysates, Smad3 proteins (phospho and nonphospho) are captured by the coated antibody. Following extensive washing, a Smad3 Mouse Detection Antibody is added to detect captured Smad3 proteins. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Smad3 proteins.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mink, Mouse

Background: Members of the Smad family of signal transduction molecules are components of a critical intracellular pathway that transmit TGF-β signals from the cell surface into the nucleus. Three distinct classes of Smads have been defined: the receptor-regulated Smads (R-Smads), which include Smad1, 2, 3, 5, and 8; the common-mediator Smad (co-Smad), Smad4; and the antagonistic or inhibitory Smads (I-Smads), Smad6 and 7 (1-5). Activated type I receptors associate with specific R-Smads and phosphorylate them on a conserved carboxy terminal SSXS motif. The phosphorylated R-Smad dissociates from the receptor and forms a heteromeric complex with the co-Smad (Smad4), allowing translocation of the complex to the nucleus. Once in the nucleus, Smads can target a variety of DNA binding proteins to regulate transcriptional responses (6-8).

$489
96 assays
1 Kit
The PathScan® Total SQSTM1/p62 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total SQSTM1/p62 protein. An SQSTM1/p62 Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, SQSTM1/p62 protein is captured by the coated antibody. Following extensive washing, SQSTM1/p62 Mouse Detection Antibody is added to detect the captured SQSTM1/p62 protein. Anti-Mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of total SQSTM1/p62 protein.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mink, Monkey, Mouse, Rat

Background: Sequestosome 1 (SQSTM1, p62) is a ubiquitin binding protein involved in cell signaling, oxidative stress, and autophagy (1-4). It was first identified as a protein that binds to the SH2 domain of p56Lck (5) and independently found to interact with PKCζ (6,7). SQSTM1 was subsequently found to interact with ubiquitin, providing a scaffold for several signaling proteins and triggering degradation of proteins through the proteasome or lysosome (8). Interaction between SQSTM1 and TRAF6 leads to the K63-linked polyubiquitination of TRAF6 and subsequent activation of the NF-κB pathway (9). Protein aggregates formed by SQSTM1 can be degraded by the autophagosome (4,10,11). SQSTM1 binds autophagosomal membrane protein LC3/Atg8, bringing SQSTM1-containing protein aggregates to the autophagosome (12). Lysosomal degradation of autophagosomes leads to a decrease in SQSTM1 levels during autophagy; conversely, autophagy inhibitors stabilize SQSTM1 levels. Studies have demonstrated a link between SQSTM1 and oxidative stress. SQSTM1 interacts with KEAP1, which is a cytoplasmic inhibitor of NRF2, a key transcription factor involved in cellular responses to oxidative stress (3). Thus, accumulation of SQSTM1 can lead to an increase in NRF2 activity.

PTMScan® Technology employs a proprietary methodology from Cell Signaling Technology (CST) for peptide enrichment by immunoprecipitation using a specific bead-conjugated antibody in conjunction with liquid chromatography (LC) tandem mass spectrometry (MS/MS) for quantitative profiling of post-translational modification (PTM) sites in cellular proteins. These include phosphorylation (PhosphoScan®), ubiquitination (UbiScan®), acetylation (AcetylScan®), and methylation (MethylScan®), among others. PTMScan® Technology enables researchers to isolate, identify, and quantitate large numbers of post-translationally modified cellular peptides with a high degree of specificity and sensitivity, providing a global overview of PTMs in cell and tissue samples without preconceived biases about where these modified sites occur. For more information on PTMScan® Proteomics Services, please visit www.cellsignal.com/common/content/content.jsp?id=ptmscan-services.
$118
10 western blots
100 µl
Nonphosphorylated 4E-BP1 Control Cell Extracts: Total cell extracts from MCF7 cells, amino acids starved for 1 hour to serve as a negative control. Supplied in SDS Sample Buffer.Phosphorylated 4E-BP1 Control Cell Extracts: Total cell extracts from MCF7 cells, amino acids starved for 1 hour followed by adding back amino acids for 1 hour and treating with 100 nM insulin for 30 min to serve as a positive control. Supplied in SDS Sample Buffer.
APPLICATIONS

Application Methods: Western Blotting

Background: Translation repressor protein 4E-BP1 (also known as PHAS-1) inhibits cap-dependent translation by binding to the translation initiation factor eIF4E. Hyperphosphorylation of 4E-BP1 disrupts this interaction and results in activation of cap-dependent translation (1). Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP1 activity (2,3). Multiple 4E-BP1 residues are phosphorylated in vivo (4). While phosphorylation by FRAP/mTOR at Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 (5).