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Antibody Sampler Kit Adrenal Gland Development

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 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 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 Nuclear Receptor Antibody Sampler Kit provides an economical means to evaluate the presence and status of nuclear receptors. This kit contains enough primary antibody to perform two western blots per primary.
The 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 PDGF Receptor β Antibody Sampler Kit provides a fast and economical means of evaluating levels of PDGF Receptor protein phosphorylated at the specified sites, as well as total PDGF receptor levels. The kit contains enough primary and secondary antibody to perform two Western blot experiments per 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).