20 results on '"Santat, Leah"'
Search Results
2. Recombinant AAV2 Transduction of Primitive Human Hematopoietic Stem Cells Capable of Serial Engraftment in Immune-Deficient Mice
- Author
-
Santat, Leah, Paz, Helicia, Wong, Christie, Li, Lijing, Macer, James, Forman, Stephen, Wong, K. K., Chatterjee, Saswati, and Berns, Kenneth I.
- Published
- 2005
3. Reconstitution of morphogen shuttling circuits.
- Author
-
Ronghui Zhu, Santat, Leah A., Markson, Joseph S., Nandagopal, Nagarajan, Gregrowicz, Jan, and Elowitz, Michael B.
- Subjects
- *
ATMOSPHERIC carbon dioxide , *BIOCHEMICAL genetics , *PHYSIOLOGIC salines , *BONE morphogenetic proteins - Abstract
The article presents a study which compared the spatiotemporal dynamics of different extracellular circuits. Topics discussed include proteins that successfully displaced gradients by shuttling ligands away from the site of production, reconstitution of morphogen and modulator gradients in vitro by sequential plating, and quantitative analysis of quasi-1 D morphogen or modulator gradients.
- Published
- 2023
- Full Text
- View/download PDF
4. A Single Lentiviral Vector Platform for MicroRNA-Based Conditional RNA Interference and Coordinated Transgene Expression
- Author
-
Shin, Kum-Joo, Wall, Estelle A., Zavzavadjian, Joelle R., Santat, Leah A., Liu, Jamie, Hwang, Jong-lk, Rebres, Robert, Roach, Tamara, Seaman, William, Simon, Melvin I., and Fraser, Iain D. C.
- Published
- 2006
- Full Text
- View/download PDF
5. Cis-interactions between Notch and Delta generate mutually exclusive signalling states
- Author
-
Sprinzak, David, Lakhanpal, Amit, LeBon, Lauren, Santat, Leah A., Fontes, Michelle E., Anderson, Graham A., Garcia-Ojalvo, Jordi, and Elowitz, Michael B.
- Subjects
Cell lines -- Research -- Physiological aspects ,Gene expression -- Research -- Physiological aspects ,T cells -- Research -- Physiological aspects ,Cellular signal transduction -- Research -- Physiological aspects ,Cell interaction -- Research -- Physiological aspects ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
The Notch-Delta signalling pathway allows communication between neighbouring cells during development (1).It has a critical role in the formation of 'fine-grained' patterns, generating distinct cell fates among groups of initially equivalent neighbouring cells and sharply delineating neighbouring regions in developing tissues (2-5).The Delta ligand has been shown to have two activities: it transactivates Notch in neighbouring cells and cis-inhibits Notch in its own cell. However, it remains unclear how Notch integrates these two activities and how the resulting system facilitates pattern formation. Here we report the development of a quantitative time-lapse microscopy platform for analysing Notch-Delta signalling dynamics in individual mammalian cells, with the aim of addressing these issues. By controlling both cis-and trans-Delta concentrations, and monitoring the dynamics of a Notch reporter, we measured the combined cis--trans input--output relationship in the Notch--Delta system. The data revealed a striking difference between the responses of Notch to trans-and cis-Delta: whereas the response to trans-Delta is graded, the response to cis-Delta is sharp and occurs at a fixed threshold, independent of trans-Delta. We developed a simple mathematical model that shows how these behaviours emerge from the mutual inactivation of Notch and Delta proteins in the same cell. This interaction generates an ultrasensitive switch between mutually exclusive sending (high Delta/low Notch) and receiving (high Notch/low Delta) signalling states. At the multicellular level, this switch can amplify small differences between neighbouring cells even without transcription-mediated feedback. This Notch-Delta signalling switch facilitates the formation of sharp boundaries and lateral-inhibition patterns in models of development, and provides insight into previously unexplained mutant behaviours., Notch and Delta are single-pass transmembrane protein families found in metazoan species. Delta in one cell can bind to, and transactivate, Notch in a neighbouring cell. This interaction results in [...]
- Published
- 2010
- Full Text
- View/download PDF
6. Cis-activation in the Notch signaling pathway.
- Author
-
Nandagopal, Nagarajan, Santat, Leah A., and Elowitz, Michael B.
- Abstract
The Notch signaling pathway consists of transmembrane ligands and receptors that can interact both within the same cell (cis) and across cell boundaries (trans). Previous work has shown that cis-interactions act to inhibit productive signaling. Here, by analyzing Notch activation in single cells while controlling cell density and ligand expression level, we show that cisligands can also activate Notch receptors. This cis-activation process resembles trans-activation in its ligand level dependence, susceptibility to cis-inhibition, and sensitivity to Fringe modification. Cisactivation occurred for multiple ligand-receptor pairs, in diverse cell types, and affected survival in neural stem cells. Finally, mathematical modeling shows how cisactivation could potentially expand the capabilities of Notch signaling, for example enabling ‘negative’ (repressive) signaling. These results establish cis-activation as an additional mode of signaling in the Notch pathway, and should contribute to a more complete understanding of how Notch signaling functions in developmental, physiological, and biomedical contexts. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
7. RNAi Methodologies for the Functional Study of Signaling Molecules.
- Author
-
Gwang Lee, Santat, Leah A., Mi Sook Chang, and Sangdun Choi
- Subjects
- *
RNA metabolism , *GENETIC transduction , *GENE silencing , *G proteins , *LABORATORY rats , *MACROPHAGE activation , *GENE expression , *CELL lines - Abstract
RNA interference (RNAi) was investigated with the aim of achieving gene silencing with diverse RNAi platforms that include small interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides (ASO). Different versions of each system were used to silence the expression of specific subunits of the heterotrimeric signal transducing G-proteins, G alpha i2 and G beta 2, in the RAW 264.7 murine macrophage cell line. The specificity of the different RNA interference (RNAi) platforms was assessed by DNA microarray analysis. Reliable RNAi methodologies against the genes of interest were then developed and applied to functional studies of signaling networks. This study demonstrates a successful knockdown of target genes and shows the potential of RNAi for use in functional studies of signaling molecules. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
8. A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs.
- Author
-
Xiaocui Zhu, Santat, Leah A., Mi Sook Chang, Liu, Jamie, Zavzavadjian, Joelle R., Wall, Estelle A., Kivork, Christine, Simon, Melvin I., and Fraser, Iain D. C.
- Subjects
- *
RNA , *GENES , *CLONING , *RETROVIRUS diseases , *PROTEIN kinases , *MAMMALS - Abstract
Background: Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems. Results: We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription. Conclusion: We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
9. A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression.
- Author
-
Kum-Joo Shin, Wall, Estelle A., Zavzavadjian, Joelle R., Santat, Leah A., Liu, Jamie, Jong-Ik Hwang, Rebres, Robert, Roach, Tamara, Seaman, William, Simon, Melvin I., and Fraser, Iain D. C.
- Subjects
RNA ,TRANSGENE expression ,ANIMAL genome mapping ,GENOMES ,GENETIC regulation ,SERUM - Abstract
RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector plat form, pSLlK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Gα12 and Gα13 both singly and in combination, demonstrating the Gα13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gβ2 correlated with a reduced Ca
2+ response to C5a. Insertion of a GFP transgene upstream of the Gβ2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLlK system. [ABSTRACT FROM AUTHOR]- Published
- 2006
- Full Text
- View/download PDF
10. Dynamic Ligand Discrimination in the Notch Signaling Pathway.
- Author
-
Nandagopal, Nagarajan, Santat, Leah A., LeBon, Lauren, Sprinzak, David, Bronner, Marianne E., and Elowitz, Michael B.
- Subjects
- *
NOTCH signaling pathway , *LIGAND analysis , *GENE expression , *GENE clusters , *ENCODING - Abstract
Summary The Notch signaling pathway comprises multiple ligands that are used in distinct biological contexts. In principle, different ligands could activate distinct target programs in signal-receiving cells, but it is unclear how such ligand discrimination could occur. Here, we show that cells use dynamics to discriminate signaling by the ligands Dll1 and Dll4 through the Notch1 receptor. Quantitative single-cell imaging revealed that Dll1 activates Notch1 in discrete, frequency-modulated pulses that specifically upregulate the Notch target gene Hes1. By contrast, Dll4 activates Notch1 in a sustained, amplitude-modulated manner that predominantly upregulates Hey1 and HeyL. Ectopic expression of Dll1 or Dll4 in chick neural crest produced opposite effects on myogenic differentiation, showing that ligand discrimination can occur in vivo . Finally, analysis of chimeric ligands suggests that ligand-receptor clustering underlies dynamic encoding of ligand identity. The ability of the pathway to utilize ligands as distinct communication channels has implications for diverse Notch-dependent processes. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
11. 19. rAAV-2 Transduction of Primitive Human Hematopoietic Stem Cells Capable of Serial Engraftment in Immune Deficient Mice
- Author
-
Paz, Helicia, Santat, Leah, Li, Lijing, Wong, Christie, Forman, Stephen J., Wong, K.K., and Chatterjee, Saswati
- Subjects
- *
GENETIC transduction - Abstract
An abstract of the article "rAAV-2 Transduction of Primitive Human Hematopoietic Stem Cells Capable of Serial Engraftment in Immune Deficient Mice," by Helicia Paz, Leah Santat, Lijing Li, Christie Wong, Stephen J. Forman, K. K. Wong and Saswati Chatterjee is presented.
- Published
- 2005
- Full Text
- View/download PDF
12. Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq.
- Author
-
Rebres, Robert A., Roach, Tamara I. A., Fraser, Iain D. C., Philip, Finly, Moon, Christina, Keng-Mean Lin, Liu, Jamie, Santat, Leah, Cheadle, Lucas, Ross, Elliott M., Simon, Melvin I., and Seaman, William E.
- Subjects
- *
MACROPHAGES , *PHOSPHOLIPASES , *RNA , *CELL receptors , *LABORATORY mice - Abstract
Cross-talk between Gαi- and Gαq-linked G-protein-coupled receptors yields synergistic Ca2+ responses in a variety of cell types. Prior studies have shown that synergistic Ca2+ responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gαq activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca2+ synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gαq. Thus, both Gβγ and Gαq contribute to activation of PLCβ3 in cells for Ca2+ synergy. We conclude that Ca2+ synergy between Gαi-coupled and Gαq-coupled receptors requires the direct action of both Gβγ and Gαq on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
13. Diversity in Notch ligand-receptor signaling interactions.
- Author
-
Kuintzle R, Santat LA, and Elowitz MB
- Abstract
The Notch signaling pathway uses families of ligands and receptors to transmit signals to nearby cells. These components are expressed in diverse combinations in different cell types, interact in a many-to-many fashion, both within the same cell (in cis) and between cells (in trans), and their interactions are modulated by Fringe glycosyltransferases. A fundamental question is how the strength of Notch signaling depends on which pathway components are expressed, at what levels, and in which cells. Here, we used a quantitative, bottom-up, cell-based approach to systematically characterize trans-activation, cis-inhibition, and cis-activation signaling efficiencies across a range of ligand and Fringe expression levels in two mammalian cell types. Each ligand (Dll1, Dll4, Jag1, and Jag2) and receptor variant (Notch1 and Notch2) analyzed here exhibited a unique profile of interactions, Fringe-dependence, and signaling outcomes. All four ligands were able to bind receptors in cis and in trans, and all ligands trans-activated both receptors, although Jag1-Notch1 signaling was substantially weaker than other ligand-receptor combinations. Cis-interactions were predominantly inhibitory, with the exception of the Dll1- and Dll4-Notch2 pairs, which exhibited cis-activation stronger than trans-activation. Lfng strengthened Delta-mediated trans-activation and weakened Jagged-mediated trans-activation for both receptors. Finally, cis-ligands showed diverse cis-inhibition strengths, which depended on the identity of the trans-ligand as well as the receptor. The map of receptor-ligand-Fringe interaction outcomes revealed here should help guide rational perturbation and control of the Notch pathway.
- Published
- 2024
- Full Text
- View/download PDF
14. Reconstitution of morphogen shuttling circuits.
- Author
-
Zhu R, Santat LA, Markson JS, Nandagopal N, Gregrowicz J, and Elowitz MB
- Subjects
- Animals, Ligands, Peptide Hydrolases, Signal Transduction, Mammals, Drosophila, Endopeptidases
- Abstract
Developing tissues form spatial patterns by establishing concentration gradients of diffusible signaling proteins called morphogens. The bone morphogenetic protein (BMP) morphogen pathway uses a family of extracellular modulators to reshape signaling gradients by actively "shuttling" ligands to different locations. It has remained unclear what circuits are sufficient to enable shuttling, what other patterns they can generate, and whether shuttling is evolutionarily conserved. Here, using a synthetic, bottom-up approach, we compared the spatiotemporal dynamics of different extracellular circuits. Three proteins-Chordin, Twsg, and the BMP-1 protease-successfully displaced gradients by shuttling ligands away from the site of production. A mathematical model explained the different spatial dynamics of this and other circuits. Last, combining mammalian and Drosophila components in the same system suggests that shuttling is a conserved capability. Together, these results reveal principles through which extracellular circuits control the spatiotemporal dynamics of morphogen signaling.
- Published
- 2023
- Full Text
- View/download PDF
15. Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q.
- Author
-
Rebres RA, Roach TI, Fraser ID, Philip F, Moon C, Lin KM, Liu J, Santat L, Cheadle L, Ross EM, Simon MI, and Seaman WE
- Subjects
- Animals, Complement C5a metabolism, Humans, Macrophages metabolism, Mice, Mice, 129 Strain, Mice, Inbred C57BL, Mutagenesis, NIH 3T3 Cells, Phospholipase C beta genetics, RNA, Small Interfering, Receptors, Purinergic P2 metabolism, Uridine Diphosphate metabolism, Calcium Signaling physiology, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, GTP-Binding Protein beta Subunits metabolism, GTP-Binding Protein gamma Subunits metabolism, Phospholipase C beta metabolism
- Abstract
Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gα(q) activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gβγ and Gα(q) contribute to activation of PLCβ3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gβγ and Gα(q) on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent.
- Published
- 2011
- Full Text
- View/download PDF
16. RNAi methodologies for the functional study of signaling molecules.
- Author
-
Lee G, Santat LA, Chang MS, and Choi S
- Subjects
- Animals, Biomedical Research methods, Cell Line, Macrophages, Methods, Mice, RNA Interference, RNA, Small Interfering, Gene Silencing, Oligonucleotide Array Sequence Analysis, Signal Transduction genetics
- Abstract
RNA interference (RNAi) was investigated with the aim of achieving gene silencing with diverse RNAi platforms that include small interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides (ASO). Different versions of each system were used to silence the expression of specific subunits of the heterotrimeric signal transducing G-proteins, G alpha i2 and G beta 2, in the RAW 264.7 murine macrophage cell line. The specificity of the different RNA interference (RNAi) platforms was assessed by DNA microarray analysis. Reliable RNAi methodologies against the genes of interest were then developed and applied to functional studies of signaling networks. This study demonstrates a successful knockdown of target genes and shows the potential of RNAi for use in functional studies of signaling molecules.
- Published
- 2009
- Full Text
- View/download PDF
17. A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs.
- Author
-
Zhu X, Santat LA, Chang MS, Liu J, Zavzavadjian JR, Wall EA, Kivork C, Simon MI, and Fraser ID
- Subjects
- Animals, Cell Adhesion Molecules metabolism, Cell Line, Cyclic AMP metabolism, Cyclic AMP pharmacology, Cyclic AMP-Dependent Protein Kinases metabolism, Genetic Vectors, Humans, Isoenzymes metabolism, Kidney cytology, Lentivirus genetics, Macrophages metabolism, Mice, Microfilament Proteins metabolism, Phosphoproteins metabolism, Phosphorylation, Plasmids, Promoter Regions, Genetic, RNA Polymerase II metabolism, RNA, Messenger metabolism, Recombination, Genetic, Retroviridae genetics, Transcription, Genetic, Transfection, Gene Silencing, MicroRNAs, RNA Interference, RNA, Small Interfering metabolism
- Abstract
Background: Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems., Results: We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription., Conclusion: We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.
- Published
- 2007
- Full Text
- View/download PDF
18. Quiescent subpopulations of human CD34-positive hematopoietic stem cells are preferred targets for stable recombinant adeno-associated virus type 2 transduction.
- Author
-
Paz H, Wong CA, Li W, Santat L, Wong KK, and Chatterjee S
- Subjects
- Bone Marrow Cells immunology, Bone Marrow Cells metabolism, Cell Cycle, Cell Proliferation, Cells, Cultured, Gene Expression, Hematopoietic Stem Cells virology, Humans, Recombinant Proteins genetics, Resting Phase, Cell Cycle, Transduction, Genetic, Transgenes, Antigens, CD34 biosynthesis, Dependovirus genetics, Genetic Therapy methods, Genetic Vectors, Hematopoietic Stem Cells immunology
- Abstract
We have previously demonstrated recombinant adeno-associated viral (rAAV) transduction of human CD34(+) hematopoietic stem cells (HSCs) capable of serial engraftment in vivo. Here we evaluated the capacity of rAAV2 to mediate gene transfer into nondividing, quiescent, primitive CD34(+) cells subdivided on the basis of metabolic, mitotic, and phenotypic properties. Results revealed that CD34(+)CD38() marrow cells are the most quiescent, exist primarily in G(0) at isolation and are the only population to remain nondividing during the entire exposure to free rAAV. Despite significant differences in the extended clonogenic capacities of CD34(+) subsets in stromal cultures, the frequency of rAAV marking of colonies derived from primitive progenitors was similar in all three populations, suggesting that both primitive and more differentiated progenitors were initially transduced at equal levels. After transduction, episomal and integrated rAAV genomes were detected in all CD34(+) subsets. However, the more quiescent cells displayed higher levels of integrated rAAV than did rapidly dividing cells. Importantly, stable long-term integration was observed only in the most primitive, quiescent CD34(+)CD38(-) subset, indicating that this HSC compartment comprises the preferred substrate for stable rAAV2 transduction. Previously described rate limitations to transgene expression were observed in transduced CD34(+) cells and could be overcome by tyrphostin pretreatment, which resulted in augmented second-strand synthesis. These results represent the first demonstration of rAAV-mediated gene transfer to primitive, quiescent human CD34(+)CD38(-) stem cells and reveal that nondividing CD34(+)CD38(-) HSCs are the optimal CD34(+) targets for rAAV transduction.
- Published
- 2007
- Full Text
- View/download PDF
19. The alliance for cellular signaling plasmid collection: a flexible resource for protein localization studies and signaling pathway analysis.
- Author
-
Zavzavadjian JR, Couture S, Park WS, Whalen J, Lyon S, Lee G, Fung E, Mi Q, Liu J, Wall E, Santat L, Dhandapani K, Kivork C, Driver A, Zhu X, Chang MS, Randhawa B, Gehrig E, Bryan H, Verghese M, Maer A, Saunders B, Ning Y, Subramaniam S, Meyer T, Simon MI, O'Rourke N, Chandy G, and Fraser ID
- Subjects
- Animals, Cell Line, Cloning, Molecular, DNA, Complementary genetics, Databases, Factual, Mice, Open Reading Frames genetics, Plasmids, Protein Kinases genetics, Signal Transduction, Protein Kinases metabolism, Proteomics
- Abstract
Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.
- Published
- 2007
- Full Text
- View/download PDF
20. The use of RNA interference to analyze protein phosphatase function in mammalian cells.
- Author
-
Fraser I, Liu W, Rebres R, Roach T, Zavzavadjian J, Santat L, Liu J, Wall E, and Mumby M
- Subjects
- Base Sequence, Blotting, Western, Cell Line, Electrophoresis, Polyacrylamide Gel, Genetic Vectors genetics, HeLa Cells, Humans, Lentivirus genetics, Molecular Sequence Data, Phosphoprotein Phosphatases genetics, RNA, Small Interfering genetics, Phosphoprotein Phosphatases metabolism, RNA Interference
- Abstract
The use of RNA interference to knock down protein phosphatases has proven to be a valuable approach to understanding the functions of these enzymes in mammalian cells. Many protein phosphatases exist as multisubunit and multigene families, which has made it difficult to assess their physiological functions using traditional approaches. The ability to selectively knock down specific subunits and individual isoforms with RNA interference has begun to make it possible to determine the contributions of individual phosphatase proteins to cellular signaling. This chapter describes methods for knocking down protein phosphatases with small interfering RNAs in easily transfectable cells and by the introduction of short-hairpin RNAs into less tractable cells using lentivirus vectors.
- Published
- 2007
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.