Description
The NanoPro 1000 system enables rapid, quantitative analysis of specific proteins in as few as 25 cells per assay. Detailed information is generated on the post-translational modification status of critical signaling proteins. The system is fully automated and speeds samples from a standard 384-well microplate to final results, ensuring exceptional sample-to-sample consistency.
The enzyme-drug, L-asparaginase, has been used since the 1970s to treat acute lymphoblastic leukemia. Asparagine synthetase (ASNS)
expression has been found to be correlated with L-asparaginase efficacy in leukemia cell lines, in leukemia primary tumor samples, and
more recently, in cancer cell lines from other tissues of origin. Silencing ASNS expression by RNAi has indicated the L-asparaginase/ASNS
relationship is causal and suggests that ASNS expression may be useful as a predictive clinical biomarker of L-asparaginase efficacy. ASNS
presents as a single peak in the NanoPro assay. Expected changes of expression are observed upon siRNA as well as L-asparaginase
treatment.
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Lactate dehydrogenase (LDH) is a reversible enzyme that catalyzes the reduction of pyruvate to lactate or the oxidation of lactate to
pyruvate. In most human cells, LDH in its native form is a tetramer with 5 possible isoforms (LDH1-5), combinations of LDHA
(calculated pI: 8.4) and/or LDHB (calculated pI: 5.7). LDH overexpression and differential expression of LDH isoforms have been
implicated in the pathogenesis and progression of many cancers.
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Translation repressor protein 4E-BP inhibits cap-dependent translation by binding to the eIF4E translation initiation factor. Hyperphosphorylation
of 4E-BP disrupts this interaction and results in activation of cap-dependent translation. Both the PI3 kinase/Akt pathway and
FRAP/mTOR kinase regulate 4E-BP activity. 4E-BP1 has been implicated as a biomarker for several cancer types, while 4E-BP2 has been
shown to potentially play a role in energy homeostasis. We show 4E-BP1 activation in MCF10A cells in response to EGF using total and
anti-phospho 4E-BP1 antibodies that enable distinction between phospho and non-phospho peaks.
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Translation repressor protein 4E-BP inhibits cap-dependent translation by binding to the eIF4E translation initiation factor. Hyperphosphorylation
of 4E-BP disrupts this interaction and results in activation of cap-dependent translation. Both the PI3 kinase/AKT pathway and
FRAP/mTOR kinase regulate 4E-BP activity. 4E-BP1 has been implicated as a biomarker for several cancer types, while 4E-BP2 has been
shown to potentially play a role in energy homeostasis. We show 4E-BP2 activation in MCF10A cells in response to EGF and 4E-BP2
inhibition in MCF7 cells with LY294002 (PI3 kinase inhibitor).
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Crk-like protein (Crk-L) is an adapter protein and phosphotyrosine-containing substrate implicated in transformation by the bcr-abl
oncogene and in signaling by cytokines. It has been shown to activate the RAS and JUN kinase signaling pathways and transform
fibroblasts in a RAS-dependent fashion. Crk-L is a substrate of the BCR-ABL tyrosine kinase and plays a role in fibroblast transformation
by BCR-ABL. We show that Crk-L phosphorylation is reduced in response to Imatinib (commonly known as Gleevec®) treatment in
K562 cells.
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Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The c-Myc-tag
consists of a short peptide sequence (MEEQKLISEEDLLM). EGFP was expressed with a c-Myc-tag at either the C- or N-terminus of the
protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed.
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Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The FLAG-tag
consists of a short peptide sequence (MADYKDDDDKM). EGFP was expressed with a FLAG-tag at either the C- or N-terminus of the
protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed.
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Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The HA-tag consists
of a short peptide sequence (MAYPYDVPDYASM). Here, EGFP was expressed with a HA-tag at both the C- and N-terminal of the
protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed.
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Dual specificity mitogen-activated protein kinases (MEK) are members of the dual specificity protein kinase family, which act upstream
from the classical MAP kinases through phosphorylation and thus activation of ERK1 and 2 in response to a wide variety of extra- and
intracellular signals. While the functions of MEK1 and MEK2 are very similar, these kinases differ significantly in the way they are regulated.
For example, serum addition can specifically induce MEK1 activity in CHO cells. By contrast, MEK2 appears to be the functionally
predominant isoform in formyl-methionyl-leucyl-phenylalanine treated neutrophils. Here we show MEK1 activation in MCF10A cells
treated with EGF.
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Dual specificity mitogen-activated protein kinases (MEK) are members of the dual specificity protein kinase family, which act upstream
from the classical MAP kinases through phosphorylation and thus activation of ERK1 and 2 in response to a wide variety of extra- and
intracellular signals. While the functions of MEK1 and MEK2 are very similar, theses kinases differ significantly in the way they are
regulated. For example, serum addition can specifically induce MEK1 activity in CHO cells. By contrast, MEK2 appears to be the functionally
predominant isoform in formyl-methionyl-leucyl-phenylalanine treated neutrophils. Here we show MEK2 activation in MCF10A cells
in response to EGF stimulation.
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