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Search results for: PhosphoSpecific

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#34913830   2021/12/16 To Up

Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27.

Cyclin-dependent kinase 4 (CDK4) is a master integrator that couples mitogenic/oncogenic signaling with the cell division cycle. It is deregulated in most cancers and inhibitors of CDK4 have become standard of care drugs for metastatic estrogen-receptor positive breast cancers and are being evaluated in a variety of other cancers. We previously characterized the T-loop phosphorylation at T172 of CDK4 as the highly regulated step that determines the activity of cyclin D-CDK4 complexes. Moreover we demonstrated that the highly variable detection of T172-phosphorylated CDK4 signals the presence or absence of the active CDK4 targeted by the CDK4/6 inhibitory drugs, which predicts the tumor cell sensitivity to these drugs including palbociclib. To date, the phosphorylation of CDK4 has been very poorly studied because only few biochemical techniques and reagents are available for it. In addition, the available ones including 2D-IEF separation of CDK4 modified forms are considered too tedious. The present report describes the generation, selection and characterization of the first monoclonal antibodies that specifically recognize the active CDK4 phosphorylated on its T172 residue. One key to this success was the immunization with a long phosphopeptide corresponding to the complete activation segment of CDK4. These monoclonal antibodies specifically recognize T172-phosphorylated CDK4 in a variety of assays, including western blotting, immunoprecipitation and, as a capture antibody, a sensitive ELISA from cell lysates. The specific immunoprecipitation of T172-phosphorylated CDK4 allowed to clarify the involvement of phosphorylations of co-immunoprecipitated p21 and p27, showing a privileged interaction of T172-phosphorylated CDK4 with S130-phosphorylated p21 and S10-phosphorylated p27. : 2D: two-dimensional; CAK: CDK-activating kinase; CDK: cyclin-dependent kinase; HAT: Hypoxanthine-Aminopterin-Thymidine; FBS: fetal bovine serum; IP: immunoprecipitation; ID: immunodetection; mAb: monoclonal antibody; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffer saline; pRb: retinoblastoma susceptibility protein; SDS: sodium dodecyl sulfate; DTT: dithiotreitol; TET: tetracyclin repressor; Avi: Avi tag; TEV: tobacco etch virus cleavage site; EGFP: enhanced green fluorescent protein; BirA: bifunctional protein biotin ligase BirA; IRES: internal ribosome entry site; HIS: poly-HIS purification tag; DELFIA: dissociation-enhanced lanthanide fluorescent immunoassay; 3-MBPP1: 1-(1,1-dimethylethyl)-3[(3-methylphenyl) methyl]-1H-pyrazolo[3,4-d] pyrimidin-4-amine; BSA: bovine serum albumin; ECL: Enhanced chemiluminescence.
Katia Coulonval, Vincent Vercruysse, Sabine Paternot, Jaime M Pita, Robert Corman, Eric Raspé, Pierre P Roger

1615 related Products with: Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27.

100 ug100 1 ml200 ug2 Pieces/Box100.00 ug200 ug200 ug200 ug1 mg200 ug

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#34884619   2021/11/26 To Up

Revisiting the Role of Ser982 Phosphorylation in Stoichiometry Shift of the Electrogenic Na/HCO Cotransporter NBCe1.

In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na-2HCO stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO influx. However, NBCe1 in the kidney mediates HCO efflux (HCO reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO efflux) than in murine colons (in which NBCe1 mediates HCO influx). Using phosphomimetic mutants of murine NBCe1 expressed in oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.
Thamer A Alsufayan, Evan J Myers, Bianca N Quade, Clayton T Brady, Aniko Marshall, Nayem Haque, Michael E Duffey, Mark D Parker

2166 related Products with: Revisiting the Role of Ser982 Phosphorylation in Stoichiometry Shift of the Electrogenic Na/HCO Cotransporter NBCe1.

11mg2 Pieces/Box1

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#34327265   2021/07/05 To Up

Visualization of Host Cell Kinase Activation by Viral Proteins Using GFP Fluorescence Complementation and Immunofluorescence Microscopy.

Non-receptor protein-tyrosine kinases regulate cellular responses to many external signals and are important drug discovery targets for cancer and infectious diseases. While many assays exist for the assessment of kinase activity , methods that report changes in tyrosine kinase activity in single cells have the potential to provide information about kinase responses at the cell population level. In this protocol, we combined bimolecular fluorescence complementation (BiFC), an established method for the assessment of protein-protein interactions, and immunofluorescence staining with phosphospecific antibodies to characterize changes in host cell tyrosine kinase activity in the presence of an HIV-1 virulence factor, Nef. Specifically, two Tec family kinases (Itk and Btk) as well as Nef were fused to complementary, non-fluorescent fragments of the Venus variant of YFP. Each kinase was expressed in 293T cells in the presence or absence of Nef and immunostained for protein expression and activity with anti-phosphotyrosine (pTyr) antibodies. Multi-color confocal microscopy revealed the interaction of Nef with each kinase (BiFC), kinase activity, and kinase protein expression. Strong BiFC signals were observed when Nef was co-expressed with both Itk and Btk, indicative of interaction, and a strong anti-pTyr immunoreactivity was also seen. The BiFC, pTyr, and kinase expression signals co-localized to the plasma membrane, consistent with Nef-mediated kinase activation in this subcellular compartment. Image analysis allowed calculation of pTyr-to-kinase protein ratios, which showed a range of responses in individual cells across the population that shifted upward in the presence of Nef and back down in the presence of a kinase inhibitor. This method has the potential to reveal changes in steady-state non-receptor tyrosine kinase activity and subcellular localization in a cell population in response to other protein-kinase interactions, information that is not attainable from immunoblotting or other methods.
Sherry T Shu, Wing Fai Li, Thomas E Smithgall

1151 related Products with: Visualization of Host Cell Kinase Activation by Viral Proteins Using GFP Fluorescence Complementation and Immunofluorescence Microscopy.

50 ug 1 kit1 kit50IU1 kit50 ug 100 ul1 kit50 ug 1mg100 ul25 Tests

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#34240493   2021/07/08 To Up

Persistent activation of the mammalian target of rapamycin signalling pathway in cutaneous squamous cell carcinomas in cats.

Cutaneous squamous cell carcinoma (CSCC) represents the most common malignant tumour of the feline skin. Emerging evidence suggests that the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signalling pathway may represent a potential target for pharmacological intervention in human and canine CSCC.
Berenice L Sanz Ressel, Adriana R Massone, Claudio G Barbeito

1812 related Products with: Persistent activation of the mammalian target of rapamycin signalling pathway in cutaneous squamous cell carcinomas in cats.

2 Pieces/Box2 Pieces/Box

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#34016897   // To Up

Blood Leukocyte Signaling Pathways as Predictors of Severity of Acute Pancreatitis.

Clinical practice lacks biomarkers to predict the severity of acute pancreatitis (AP). We studied if intracellular signaling of circulating leukocytes could predict persistent organ dysfunction (OD) and secondary infections in AP.
Antti Turunen, Antti Kuuliala, Harri Mustonen, Pauli Puolakkainen, Leena Kylänpää, Krista Kuuliala

1078 related Products with: Blood Leukocyte Signaling Pathways as Predictors of Severity of Acute Pancreatitis.

Unit 5 G0.5 mg9 x 25 assays100ug Lyophilized1 ST250200 assays400 Assays

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#33498635   2021/01/23 To Up

Differential Role of Threonine and Tyrosine Phosphorylation in the Activation and Activity of the Yeast MAPK Slt2.

The Mitogen-Activated Protein Kinase (MAPK) Slt2 is central to signaling through the yeast Cell Wall Integrity (CWI) pathway. MAPKs are regulated by phosphorylation at both the threonine and tyrosine of the conserved TXY motif within the activation loop (T190/Y192 in Slt2). Since phosphorylation at both sites results in the full activation of MAPKs, signaling through MAPK pathways is monitored with antibodies that detect dually phosphorylated forms. However, most of these antibodies also recognize monophosphorylated species, whose relative abundance and functionality are diverse. By using different phosphospecific antibodies and phosphate-affinity (Phos-tag) analysis on distinct Slt2 mutants, we determined that Y192- and T190-monophosphorylated species coexist with biphosphorylated Slt2, although most of the Slt2 pool remains unphosphorylated following stress. Among the monophosphorylated forms, only T190 exhibited biological activity. Upon stimulation, Slt2 is first phosphorylated at Y192, mainly by the MAPKK Mkk1, and this phosphorylation is important for the subsequent T190 phosphorylation. Similarly, dephosphorylation of Slt2 by the Dual Specificity Phosphatase (DSP) Msg5 is ordered, with dephosphorylation of T190 depending on previous Y192 dephosphorylation. Whereas Y192 phosphorylation enhances the Slt2 catalytic activity, T190 is essential for this activity. The conserved T195 residue is also critical for Slt2 functionality. Mutations that abolish the activity of Slt2 result in a high increase in inactive Y192-monophosphorylated Slt2. The coexistence of different Slt2 phosphoforms with diverse biological significance highlights the importance of the precise detection of the Slt2 phosphorylation status.
Gema González-Rubio, Ángela Sellers-Moya, Humberto Martín, María Molina

2845 related Products with: Differential Role of Threonine and Tyrosine Phosphorylation in the Activation and Activity of the Yeast MAPK Slt2.

2 Pieces/Box2 Pieces/Box2 Pieces/Box1

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#33452816   2021/02/28 To Up

The endolysosomal adaptor PLEKHM1 is a direct target for both mTOR and MAPK pathways.

The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain-containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 coprecipitates with, and is directly phosphorylated by, mTOR. Using a phosphospecific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2-mediated phosphorylation in a growth factor-dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor- and amino acid-sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism.
Andrea Gubas, Christina Karantanou, Doris Popovic, Georg Tascher, Marina E Hoffmann, Anna Platzek, Nina Dawe, Ivan Dikic, Daniela S Krause, David G McEwan

1529 related Products with: The endolysosomal adaptor PLEKHM1 is a direct target for both mTOR and MAPK pathways.

25 µg250 ml0.25 mg100 TESTS0.1 mg1 LITRE0.1 ml0.1 mg0.1ml (1mg/ml)100 ml0.2 mg1 ml

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