Epitope Tag-Antibody Combination Useful for the Detection of Protein Expression in Prokaryotic and Eukaryotic Cells

Rüdiger, Manfred; Jockusch, Brigitte M.; Rothkegel, Martin

doi:10.2144/97231bm20

Cited by 32 publications

(40 citation statements)

References 4 publications

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“…Bovine PKC␣ cDNA (a gift of Dr. Peter J. Parker, Imperial Cancer Research Fund, London) was equipped with an N-terminal birch-profilin epitope tag (35) and cloned into the expression vector pcDNA3 (Invitrogen). An ATP binding-deficient mutant of PKC␣ (K368M, Ref.…”

Section: Methodsmentioning

confidence: 99%

“…Excess cross-linker was quenched in 0.2 M glycine in phosphate-buffered saline, and cells were lysed in radioimmune precipitation assay buffer (50 mM Tris, pH 7.2, 1% (v/v) Triton X-100, 0.25% deoxycholate, 1 mM EGTA, 150 mM NaCl, protease inhibitors). Following adsorption to protein G-Sepharose beads (Amersham Biosciences), precleared lysates were subjected to immunoprecipitation with BiPro tag antibody (35) or FLAG M2 antibody (Sigma). Bound protein complexes were eluted from beads by boiling in SDS sample buffer, separated by SDS-PAGE, and transferred onto nitrocellulose membrane.…”

Section: Methodsmentioning

confidence: 99%

“…After extensive washing in TBST, bound proteins were detected by autoradiography and quantified by phosphorimaging (Fuji BAS-2500) using Aida 2.1 (Advanced Isotope Data Analyser) analysis software (Fuji; Raytest, Straubenhardt, Germany). Means of bound protein, corrected for [ 35 S]methionine labeling of PKC␣ (rd (8 met), V3 (0 met), kd (12 met)) from four independent experiments were compared by variance analysis (Scheffe, p Ͻ 0.05) using Statview 5.0 (SAS Institute Inc., Cary, NC).…”

Section: Methodsmentioning

confidence: 99%

“…Standard culture conditions result in a basal activation of PKC, which is thought to involve transient membrane localization of the kinase and interaction with different protein ligands (42). FLN fragments were equipped with a FLAG tag (35) and subcloned in expression vectors (pcDNA3, Invitrogen) to test their interaction with PKC␣, after co-transfection in HeLa cells. A membrane permeant cross-linker was applied prior to lysis of cells to stabilize PKC-ligand interactions.…”

Section: Methodsmentioning

confidence: 99%

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The F-actin Cross-linking and Focal Adhesion Protein Filamin A Is a Ligand and in Vivo Substrate for Protein Kinase Cα

Tigges

Koch

Wissing

et al. 2003

Journal of Biological Chemistry

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Filamin A is an established structural component of cell-matrix adhesion sites. In addition, it serves as a scaffold for the subcellular targeting of different signaling molecules. Protein kinase C (PKC) has been found associated with filamin; however, details about this interaction and its significance for cell-matrix adhesiondependent signaling have remained elusive. We performed a yeast two-hybrid analysis using protein kinase C␣ as a bait and identified filamin as a direct binding partner. The interaction was confirmed in transfected HeLa cells, and serial truncation fragments of filamin A were employed to identify two binding sites on filamin. In vitro ligand binding assays revealed a Ca 2؉ and phospholipid-dependent association of the regulatory domain of protein kinase C with these sites. Phosphorylation of filamin was found to be isoform-restricted, leading to phosphate incorporation in the C termini of filamin A and C, but not B. PKC-dependent phosphorylation of filamin was also detected in cells. Our data suggest an intimate interaction between filamin and PKC in cell signaling.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The F-actin Cross-linking and Focal Adhesion Protein Filamin A Is a Ligand and in Vivo Substrate for Protein Kinase Cα

Tigges

Koch

Wissing

et al. 2003

Journal of Biological Chemistry

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“…EcadM was generated by adding the myristylation sequence (MGSSKSKPKDPSQR) derived from v-Src as described [28]. EcadMOM was generated by adding a mitochondrial outer membrane targeting sequence [29] to the N-terminus of EcadCD. A chimeric construct comprised of the extracellular domain of N-cadherin and the cytoplasmic domain of E-cadherin has been described [22].…”

Section: Constructs Transfections and Infectionsmentioning

confidence: 99%

The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane

Fukumoto

Shintani

Reynolds

et al. 2008

Experimental Cell Research

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In contrast to growth factor-stimulated tyrosine phosphorylation of p120, its relatively constitutive serine/threonine phosphorylation is not well understood. Here we examined the role of serine/ threonine phosphorylation of p120 in cadherin function. Expression of cadherins in cadherin-null cells converted them to an epithelial phenotype, induced p120 phosphorylation and localized it to sites of cell contact. Detergent solubility and immunofluorescence confirmed that phosphorylated p120 was at the plasma membrane. E-cadherin constructs incapable of traveling to the plasma membrane did not induce serine/threonine phosphorylation of p120, nor did cadherins constructs incapable of binding p120. However, an E-cadherin cytoplasmic domain construct artificially targeted to the plasma membrane did induce serine/threonine phosphorylation of p120, suggesting phosphorylation occurs independently of signals from cadherin dimerization and trafficking through the ER/Golgi. Solubility assays following calcium switch showed that p120 isoform 3A was more effective at stabilizing E-cadherin at the plasma membrane relative to isoform 4A. Since the major phosphorylation domain of p120 is included in isoform 3A but not 4A, we tested p120 mutated in the known phosphorylation sites in this domain, and found that it was even less effective at stabilizing E-cadherin. These data suggest that serine/threonine phosphorylation of p120 influences the dynamics of E-cadherin in junctions.

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Raver2, a new member of the hnRNP family

et al. 2005

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Raver2 was identified as a novel member of the hnRNP family based on sequence homology within three RNA recognition motifs and its general domain organization reminiscent of the previously described raver1 protein. Like raver1, raver2 contains two putative nuclear localization signals and a potential nuclear export sequence, and also displays nucleocytoplasmic shuttling in a heterokaryon assay. In glia cells and neurons, raver2 localizes to the nucleus. Moreover, the protein interacts with polypyrimidine tract binding protein (PTB) suggesting that it may participate in PTB-mediated nuclear functions. In contrast to ubiquitously expressed raver1, raver2 exerts a distinct spatio-temporal expression pattern during embryogenesis and is essentially restricted to brain, lung, and kidney in the adult mouse.

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Epitope Tag-Antibody Combination Useful for the Detection of Protein Expression in Prokaryotic and Eukaryotic Cells

Cited by 32 publications

References 4 publications

The F-actin Cross-linking and Focal Adhesion Protein Filamin A Is a Ligand and in Vivo Substrate for Protein Kinase Cα

The F-actin Cross-linking and Focal Adhesion Protein Filamin A Is a Ligand and in Vivo Substrate for Protein Kinase Cα

The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane

Raver2, a new member of the hnRNP family

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