Interested in promotions? | Click here >>

Antibody Sampler Kit Cytoskeleton-Dependent Intracellular Transport

The Loading Control Antibody Sampler Kit provides an economical means to detect a variety of housekeeping proteins. The kit contains enough primary and secondary antibodies to perform two western blot experiments.
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 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 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 Insulin/IGF-1 Signaling Pathway Antibody Sampler Kit provides an economical means of detecting select components involved in the insulin and/or IGF-1 signaling pathways. The kit contains enough primary antibodies to perform at least two western blot experiments per antibody.

Background: Insulin and IGF-1 act on two closely related tyrosine kinase receptors to initiate a cascade of signaling events. These signaling events activate a variety of biological molecules, including kinases and transcription factors, which regulate cell growth, survival and metabolism.Type I insulin-like growth factor receptor (IGF-IR) is a transmembrane receptor tyrosine kinase that is widely expressed in many cell lines and cell types within fetal and postnatal tissues (1-3). Three tyrosine residues within the kinase domain (Tyr1131, Tyr1135, and Tyr1136) are the earliest major autophosphorylation sites (4). Phosphorylation of these three tyrosine residues is necessary for kinase activation (5,6). Insulin receptors (IRs) share significant structural and functional similarity with IGF-I receptors, including the presence of an equivalent tyrosine cluster (Tyr1146/1150/1151) within the kinase domain activation loop. Tyrosine autophosphorylation of IRs is one of the earliest cellular responses to insulin stimulation (7). Autophosphorylation begins with phosphorylation at Tyr1146 and either Tyr1150 or Tyr1151, while full kinase activation requires triple tyrosine phosphorylation (8).Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (9-11). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (10,11). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (12) 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 (13,14).Tuberin is a product of the TSC2 tumor suppressor gene and an important regulator of cell proliferation and tumor development (15). Tuberin is phosphorylated on Ser939 and Thr1462 in response to PI3K activation and the human TSC complex is a direct biochemical target of the PI3K/Akt pathway (16). This result complements Drosophila genetics studies suggesting the possible involvement of the tuberin-hamartin complex in the PI3K/Akt mediated insulin pathway (17-19).The mammalian target of rapamycin (mTOR, FRAP, RAFT) is a Ser/Thr protein kinase (20-22) that functions as an ATP and amino acid sensor to balance nutrient availability and cell growth (23,24). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal to transmit a positive signal to p70 S6 kinase and participate in the inactivation of the eIF4E inhibitor, 4E-BP1 (25). These events result in the translation of specific mRNA subpopulations. mTOR is phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway and autophosphorylated at Ser2481 (26,27).The Forkhead family of transcription factors is involved in tumorigenesis of rhabdomyosarcoma and acute leukemias (28-30). Within the family, three members (FoxO1, FoxO4, and FoxO3a) have sequence similarity to the nematode orthologue DAF-16, which mediates signaling via a pathway involving IGFR1, PI3K, and Akt (31-33). Active forkhead members act as tumor suppressors by promoting cell cycle arrest and apoptosis. Increased proliferation results when forkhead transcription factors are inactivated through phosphorylation by Akt at Thr24, Ser256, and Ser319, which results in nuclear export and inhibition of transcription factor activity (34).Glycogen synthase kinase-3 (GSK-3) was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (35). 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 (36,37).

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

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