scholarly journals Carboxymethylation of nuclear protein serine/threonine phosphatase X

1997 ◽  
Vol 327 (2) ◽  
pp. 481-486 ◽  
Author(s):  
Susanne KLOEKER ◽  
C. Jeffrey BRYANT ◽  
Stefan STRACK ◽  
J. Roger COLBRAN ◽  
E. Brian WADZINSKI
Keyword(s):  
Polyclonal Antibodies ◽  
Alkali Treatment ◽  
Homologous Protein ◽  
Tissue Extracts ◽  
Cell Extracts ◽  
Nuclear Extracts ◽  
C Terminus ◽  
Phosphatase 2A ◽  
Internal Peptide ◽  
Rat Tissue

Specific rabbit polyclonal antibodies against peptides corresponding to the highly homologous protein serine/threonine phosphatase 2A and X catalytic subunits (PP2A/C and PPX/C respectively) were used to investigate the cellular and subcellular distribution of PP2A/C and PPX/C, as well as their methylation state. Immunoblots of rat tissue extracts revealed a widespread distribution of these enzymes but particularly high levels of PP2A/C and PPX/C in brain and testes respectively. In addition, immunoblots of subcellular fractions and immunocytochemical analyses of rat brain sections demonstrated that PPX/C is predominantly localized to the nucleus, whereas PP2A/C is largely cytoplasmic. Treatment of nuclear extracts with alkali resulted in increased PPX/C immunoreactivity to a polyclonal antibody directed against the C-terminus; no change in PPX immunoreactivity was observed using an antibody against an internal peptide. Alkali treatment of brain and liver cytosolic and nuclear extracts did not change the molecular mass or the isoelectric point of PPX/C. Furthermore, tritiated PPX/C was immunoprecipitated from COS cell extracts incubated with the methyl donor S-adenosyl-l-[methyl-3H]methionine. Thus the increase in immunoreactivity probably results from removal of a carboxymethyl group from PPX/C, as has been shown previously for PP2A/C [Favre, Zolnierowicz, Turowski and Hemmings (1994) J. Biol. Chem. 269, 16311-16317]. Together, our results indicate that the PPX catalytic subunit is a predominantly nuclear phosphatase and is methylated at its C-terminus.

Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit

1994 ◽  
Vol 14 (12) ◽  
pp. 8183-8190
Author(s):  
A Jenny ◽  
H P Hauri ◽  
W Keller
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Tryptic Digest ◽  
Cell Extracts ◽  
C Terminus ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Precursor Rna ◽  
Novel Protein

During the formation of the 3' ends of mRNA, the cleavage and polyadenylation specificity factor (CPSF) is required for 3' cleavage of the transcript as well as for subsequent polyadenylation. Using peptide sequences from a tryptic digest, we have cloned the 100-kDa subunit of CPSF. This subunit is a novel protein showing no homology to any known polypeptide in databases. Polyclonal antibodies against the C terminus of the protein inhibit the polyadenylation reaction. Polyclonal and monoclonal antibodies were used to characterize the composition of CPSF. Immunoprecipitations of CPSF from HeLa cell extracts and from labeled chromatographic fractions show the coprecipitation of all four subunits of 160, 100, 73, and 30 kDa. Proteins of 160 and 30 kDa that are specifically cross-linked to precursor RNA by UV irradiation were identified as CPSF subunits by immunoprecipitation. Immunofluorescent detection of CPSF in HeLa cells localized it in the nucleoplasm, excluding cytoplasm and nucleolar structures.

scholarly journals Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit.

1994 ◽  
Vol 14 (12) ◽  
pp. 8183-8190 ◽  
Author(s):  
A Jenny ◽  
H P Hauri ◽  
W Keller
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Tryptic Digest ◽  
Cell Extracts ◽  
C Terminus ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Precursor Rna ◽  
Novel Protein

During the formation of the 3' ends of mRNA, the cleavage and polyadenylation specificity factor (CPSF) is required for 3' cleavage of the transcript as well as for subsequent polyadenylation. Using peptide sequences from a tryptic digest, we have cloned the 100-kDa subunit of CPSF. This subunit is a novel protein showing no homology to any known polypeptide in databases. Polyclonal antibodies against the C terminus of the protein inhibit the polyadenylation reaction. Polyclonal and monoclonal antibodies were used to characterize the composition of CPSF. Immunoprecipitations of CPSF from HeLa cell extracts and from labeled chromatographic fractions show the coprecipitation of all four subunits of 160, 100, 73, and 30 kDa. Proteins of 160 and 30 kDa that are specifically cross-linked to precursor RNA by UV irradiation were identified as CPSF subunits by immunoprecipitation. Immunofluorescent detection of CPSF in HeLa cells localized it in the nucleoplasm, excluding cytoplasm and nucleolar structures.

scholarly journals Augmentation of ACTH-induced steroidogenesis in isolated rat adrenal cells by rat tissue extracts and by hormone preparations.

1982 ◽  
Vol 29 (2) ◽  
pp. 251-254 ◽  
Author(s):  
SAYOMI IIDA ◽  
KEIKO MATSUYAMA ◽  
YOSHIHARU ITOH ◽  
KANAME MORIWAKI ◽  
SEIICHIRO TARUI ◽  
...  
Keyword(s):  
Adrenal Cells ◽  
Tissue Extracts ◽  
Rat Tissue ◽  
Isolated Rat

Workflow for proteomic analysis of purified lysosomes with or without damage v2

2021 ◽  
Author(s):  
Sharan Swarup ◽  
J. Wade Harper
Keyword(s):  
Proteomic Analysis ◽  
Quantitative Proteomics ◽  
Eukaryotic Cells ◽  
Ester Hydrochloride ◽  
Methyl Ester Hydrochloride ◽  
Metabolomic Analysis ◽  
Epitope Tag ◽  
Cell Extracts ◽  
C Terminus ◽  
Processing Steps

Lysosomes are a major degradative organelle within eukaryotic cells. Previous work has developed a method wherein the TMEM192 protein is tagged on its C-terminus with an epitope tag in order to immunopurify (IP) lysosomes from cell extracts.1 This process is referred to as Lyso-IP. Such lysosomes can be used for proteomic analysis or for metabolomic analysis. The Lyso-IP is adapted from a previous reported method (Wyant et al., 2018). Here we also describe processing steps using proteomics after lysosome purification in the context of lysosomal damaging agents. Agents such as L-Leucyl-L-Leucine methyl ester (hydrochloride) (LLoMe) and Gly-Phe-β-naphthylamide (GPN) induce lysosomal damage, leading to the degradation of damaged lysosomes by lysophagy. This adaptation of Lyso-IP provides a route to identify proteins that are recruited to damaged lysosomes using quantitative proteomics.

scholarly journals A simple method to measure CLOCK-BMAL1 DNA binding activity in tissue and cell extracts

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1316 ◽  
Author(s):  
Maud Gillessen ◽  
Pieter Bas Kwak ◽  
Alfred Tamayo
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Casein Kinase I ◽  
Simple Method ◽  
Dna Binding Activity ◽  
Tissue Extracts ◽  
Cell Extracts ◽  
Specialized Equipment ◽  
Dna Binding Assay

The proteins CLOCK and BMAL1 form a heterodimeric transcription factor essential to circadian rhythms in mammals.  Daily rhythms of CLOCK-BMAL1 DNA binding activity are known to oscillate with target gene expression in vivo. Here we present a highly sensitive assay that recapitulates native CLOCK-BMAL1 DNA binding rhythms from crude tissue extracts, which we call the Clock Protein-DNA Binding Assay (CPDBA). This method can detect less than 2-fold differences in DNA binding activity, and can deliver results in two hours or less using 10 microliters or less of crude extract, while requiring neither specialized equipment nor expensive probes. To demonstrate the sensitivity and versatility of this assay, we show that enzymatic removal of phosphate groups from proteins in tissue extracts or pharmacological inhibition of casein kinase I in cell culture increased CLOCK-BMAL1 DNA binding activity by ~1.5 to ~2 fold, as measured by the CPDBA. In addition, we show that the CPDBA can measure CLOCK-BMAL1 binding to reconstituted chromatin. The CPDBA is a sensitive, fast, efficient and versatile probe of clock function.

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts

1985 ◽  
Vol 5 (12) ◽  
pp. 3331-3336
Author(s):  
K Y Song ◽  
L Chekuri ◽  
S Rauth ◽  
S Ehrlich ◽  
R Kucherlapati
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Double Strand Breaks ◽  
Base Pairs ◽  
In Vitro System ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

scholarly journals A Novel Tissue and Stem Cell Specific TERF1 Splice Variant Is Downregulated in Tumour Cells

2019 ◽  
Vol 21 (1) ◽  
pp. 85
Author(s):  
Yousef Ashraf Tawfik Morcos ◽  
Gregoire Najjar ◽  
Sabine Meessen ◽  
Britta Witt ◽  
Anca Azoitei ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Splice Variant ◽  
Polyclonal Antibodies ◽  
Genome Comparison ◽  
Rna Expression ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
C Terminus ◽  
The Impact

In this study, we describe the identification of a novel splice variant of TERF1/PIN2, one of the main components of the telomeric shelterin complex. This new splice variant is identical to TERF1, apart from a 30 amino acid internal insertion near to the C-terminus of TERF1. Based on genome comparison analyses and RNA expression data, we show that this splice variant is conserved among hominidae but absent from all other species. RNA expression and histological analyses show specific expression in human spermatogonial and hematopoietic stem cells (HSCs), while all other analyzed tissues lack the expression of this TERF1-isoform, hence the name TERF1-tsi (TERF1-tissue-specific-isoform). In addition, we could not detect any expression in primary human cells and established cancer cell lines. Immunohistochemistry results involving two new rabbit polyclonal antibodies, generated against TERF1-tsi specific peptides, indicate nuclear localization of TERF1-tsi in a subset of spermatogonial stem cells. In line with this observation, immunofluorescence analyzes in various cell lines consistently revealed that ectopic TERF1-tsi localizes to the cell nucleus, mainly but not exclusively at telomeres. In a first attempt to evaluate the impact of TERF1-tsi in the testis, we have tested its expression in normal testis samples versus matched tumor samples from the same patients. Both RT-PCR and IHC show a specific downregulation of TERF1-tsi in tumor samples while the expression of TERF1 and PIN2 remains unchanged.

scholarly journals The 72/74-kDa polypeptides of the 70–110S large heterogeneous nuclear ribonucleoprotein complex (LH-nRNP) represent a discrete subset of the hnRNP M protein family

2000 ◽  
Vol 350 (2) ◽  
pp. 495-503 ◽  
Author(s):  
Panayiota KAFASLA ◽  
Meropi PATRINOU-GEORGOULA ◽  
Apostolia GUIALIS
Keyword(s):  
Matrix Protein ◽  
Polyclonal Antibodies ◽  
Protein Family ◽  
M Protein ◽  
Major Protein ◽  
Discrete Subset ◽  
Two Dimensional Gel Electrophoresis ◽  
Nuclear Extracts ◽  
Protein Components ◽  
Nuclear Ribonucleoprotein

Pre-mRNA processing in eukaryotes is thought to take place on a multitude of nuclear ribonucleoprotein (RNP) complexes, the most abundant of them being the heterogeneous nuclear (hn) RNP complexes. The identification in mammalian nuclear extracts of a novel, less-abundant 70–110S heterogeneous RNP, named large heterogeneous nuclear RNP (LH-nRNP), has previously been reported by Aidinis, Sekeris and Guialis (1995) Nucleic Acids Res. 23, 2742–2753. The structural composition of the LH-nRNP complex has been determined following the production of polyclonal antibodies against the major protein constituents of the complex, the pair of the 72/74-kDa polypeptides. In the present study evidence is shown to prove that the 72/74-kDa proteins are members of the hnRNP M protein family, hereafter referred to as 72/74(M) polypeptides. The extensive application of two-dimensional gel electrophoresis, combined with specific immunoprecipitation and immunoblotting assays, has allowed the assignment of the 72/74(M) proteins to a subset of the hnRNP M family, characteristic of the presence of the LH-nRNP complex and distinct from the hnRNP-associated M1–M4 components. Moreover, the immunoselection of the LH-nRNP complex from [32P]orthophosphate-labelled HeLa cells, with the parallel application of UV irradiation, has permitted the identification of the 72/74(M) polypeptides as the sole protein constituents of the complex in direct contact with the RNA. It is proposed that LH-nRNP constitutes a discrete subset of hnRNP complexes, having a possible role in establishing specific interactions between hnRNP and nuclear-matrix protein components.

scholarly journals Human Kallikrein 13: Production and Purification of Recombinant Protein and Monoclonal and Polyclonal Antibodies, and Development of a Sensitive and Specific Immunofluorometric Assay

2003 ◽  
Vol 49 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Carl Kapadia ◽  
Albert Chang ◽  
Georgia Sotiropoulou ◽  
George M Yousef ◽  
Linda Grass ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Amniotic Fluid ◽  
Polyclonal Antibodies ◽  
Biological Fluids ◽  
Expression System ◽  
Immunohistochemical Analysis ◽  
Cross Reactivity ◽  
Yeast Expression System ◽  
Tissue Extracts ◽  
Cancer Tissues

Abstract Background: The aims of this study were to develop immunologic reagents and a sensitive and specific immunoassay for human kallikrein 13 (hK13) and to examine the presence of hK13 in human tissues and biological fluids. Methods: Recombinant hK13 protein was produced and purified with use of a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse monoclonal and rabbit polyclonal anti-hK13 antibodies. A sandwich-type immunoassay was developed with these antibodies. The assay was used to measure hK13 in various biological fluids and tissue extracts. Immunohistochemical analysis was also performed on nondiseased and cancerous prostatic sections. Results: The hK13 immunoassay had a detection limit of 0.05 μg/L and showed no cross-reactivity with homologous kallikreins. The assay was linear at 0–20 μg/L, and within-and between-run CVs were <10% (n = 12). hK13 was detected in tissues, including esophagus, tonsil, trachea, lung, cervix, and prostate. hK13 was also found in seminal plasma, amniotic fluid, follicular fluid, ascites of ovarian cancer patients, breast milk, and cytosolic extracts of ovarian cancer tissues. hK13 was immunohistochemically localized in epithelial cells of both nondiseased and cancerous prostate. hK13 appears to be overexpressed in 50% of ovarian cancer tissues compared with healthy ovarian tissues. Recovery of active enzyme added to milk or amniotic fluid was 70–98%, but was <20% when added to serum, suggesting rapid sequestration by protease inhibitors. In fluids and tissue extracts, hK13 was found in its free (∼30 kDa) form. Conclusions: This immunofluorometric assay for hK13 may be used to examine the value of hK13 as a disease biomarker and to further explore the physiologic and pathobiologic role of this enzyme in human disease.

scholarly journals Retinoic acid receptor gamma: specific immunodetection and phosphorylation.

1991 ◽  
Vol 115 (2) ◽  
pp. 535-545 ◽  
Author(s):  
C Rochette-Egly ◽  
Y Lutz ◽  
M Saunders ◽  
I Scheuer ◽  
M P Gaub ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Polyclonal Antibodies ◽  
Retinoic Acid Receptor ◽  
Amino Acid Sequences ◽  
Expression Vectors ◽  
Acid Receptor ◽  
F Region ◽  
Nuclear Extracts ◽  
F9 Embryonal Carcinoma Cells ◽  
Human And Mouse

Synthetic peptides corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid receptor gamma 1 (hRAR-gamma 1 and mRAR-gamma 1, respectively) were used to generate anti-RAR-gamma 1 antibodies. Four mAbs were selected, which were directed against peptides found in region A1 (Ab1 gamma (A1)), region F (Ab2 gamma (mF) and Ab4 gamma (hF)) and region D2 (Ab5 gamma (D2)). These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-gamma 1 proteins in COS-1 cells transfected with expression vectors containing the RAR-gamma 1 cDNAs. They all reacted with both human and mouse RAR-gamma 1 proteins, except Ab4 gamma (hF) that was specific for hRAR-gamma 1. Rabbit polyclonal antibodies, directed against a peptide from the mRAR-gamma 1 F region were also obtained (RP gamma (mF)) and found to be specific for mouse RAR-gamma 1 protein. Furthermore, in gel retardation/shift assays the antibodies specifically retarded the migration of complexes obtained with a RA response element (RARE). Antibodies raised against regions D2 and F also recognized the RAR-gamma 2 isoform which differs from RAR-gamma 1 only in the A region. On the other hand, antibodies directed against the A1 region of RAR-gamma 1 (Ab1 gamma (A1)) only reacted with the RAR-gamma 1 protein. The antibodies characterized here allowed us to detect the presence of mRAR-gamma 1 and gamma 2 isoforms in mouse embryos and F9 embryonal carcinoma cells nuclear extracts. They were also used to demonstrate that the mRAR-gamma 1 protein can be phosphorylated and that the phosphorylation occurs mainly in the NH2-terminal A/B region.

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