Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets

Glass, William F.; Briggs, Robert C.; Hnilica, Lubomir S.

doi:10.1016/0003-2697(81)90549-2

Cited by 231 publications

(76 citation statements)

References 10 publications

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“…Previously it has been shown that specific subclasses of nuclear-envelope-associated proteins can be recognized by lectins (7,(12)(13)(14)17). In the present study, we used fluorescently labeled lectins to characterize preparations of isolated nuclei.…”

Section: Resultsmentioning

confidence: 99%

Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

Markland¹,

Smith²,

Roberts³

1987

Mol. Cell. Biol.

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An in vitro nuclear translocation system is described in which isolated rat liver nuclei were incubated in a defined buffered medium containing radiolabeled or fluorescently labeled exogenous proteins. The nuclei were rapidly recovered, extracted, and analyzed for the presence of associated radiolabeled or fluorescently labeled proteins. The isolated nuclei exhibited the same specificity for protein uptake as seen previously in vivo, accumulating simian virus 40 wild-type large-T antigen and p53 while excluding a cytoplasmic variant of large-T antigen (dlO) and bovine serum albumin. The rapid nuclear accumulation of wild-type large-T antigen was shown to be selective and dependent upon the recognition of a wild-type nuclear location signal, ATP and temperature dependent, and unidirectional. Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process.A nuclear location signal sequence has been identified in simian virus 40 (SV40) large-T antigen (18). The sequence Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu is both necessary and sufficient for the nuclear localization of large-T antigen variants as well as otherwise cytoplasmic proteins (19). Synthetic peptides homologous to the SV40 large-T antigen nuclear signal can also target proteins of various sizes to the nucleus (15,22).Several known nuclear proteins contain basic amino acid sequences homologous to the SV40 Iarge-T antigen prototypic signal sequence (36). In some cases, it has been shown that these sequences constitute the putative nuclear location signals of their respective proteins (32, 39). The large-T antigen of polyomavirus contains two basic sequences which contribute to the nuclear accumulation of this protein (30). Recently, it has been demonstrated that the efficacy of a nuclear location signal is dependent upon the context of the protein within which it is present (33).Little is known about how nuclear location signals function. The SV40 large-T antigen signal permits the rapid nuclear entry of proteins which would otherwise be too large to accumulate in the nucleus by passive diffusion (15,19,22). This process exhibits a high degree of specificity, since a single point mutation at codon 128 of the signal sequence can completely abolish this phenomenon (5,18,21). This suggests that a highly efficient and selective cellular apparatus must be responsible for the recognition and nuclear translocation of proteins containing nuclear location signals.The development of a cell-free system to study the signaldependent nuclear accumulation of proteins would facilitate the characterization of nuclear signal-mediated translocation of proteins across the nuclear envelope and the identification of the translocation apparatus itself. The inclusion of homogeneous preparations of isolated nuclei in such a system would enhance the likelihood that it would reproducibly exhibit nuclear transport characteristics similar to those observed previously in vi...

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Section: Resultsmentioning

confidence: 99%

Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

Markland¹,

Smith²,

Roberts³

1987

Mol. Cell. Biol.

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“…Electroblotting on nitrocellulose membranes (Biotrace NT; Gelman Science, Ann Arbor, MI) was in accordance with standard procedures. 36 Each blot was then treated with 5% Gloria milk (Nestlé, Vévey, Switzerland) with exception for phosphorylation of ␤-catenin (3% BSA), in trisbuffered saline (TBS) for 4 hr prior to overnight incubation with MAb 214D4 for episialin, HECD-1 for E-cadherin and anti-␤-catenin for ␤-catenin and PY20 (ICN, Irvine, CA) for phosphorylation of ␤-catenin. After washing, blots were incubated for 2 hr with horseradish peroxidase-conjugated secondary antibody.…”

Section: Western Blotting Of Episialin and E-cadherinmentioning

confidence: 99%

Alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl- glycerophosphocholine induces invasion through episialin-mediated neutralization of E-cadherin in human mammary MCF-7 cellsin vitro

et al. 2001

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1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18 Key words: alkyl-lysophospholipid; E-cadherin; episialin; adhesion; invasion1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OMe) interacts with cell membranes, thereby modulating phospholipid metabolism, interfering with signal transduction, inducing apoptosis and differentiation and regulating invasion. 1 On solid substrate, the E-cadherin/catenin complex is implicated in the cell-cell adhesion of 2 variants of the MCF-7 human breast cancer cell line family, coined MCF-7/AZ and MCF-7/6. In suspension, however, MCF-7/6 cells are adhesion-deficient while MCF-7/AZ cells are adhesion-proficient. Treatment with ET-18-OMe caused antithetic effects on the 2 types of MCF-7 cells. ET-18-OMe restored the E-cadherin function in the adhesiondeficient MCF-7/6 cells, whereas it caused a decrease in cellular aggregation of the adhesion-proficient MCF-7/AZ cells. The latter effects could not be explained by changes in sialylation of Ecadherin. 2 Antithetic effects on invasion have also been reported: ET-18-OMe inhibited invasion of constitutively invasive cells, [3][4][5][6] whereas it induced invasion of otherwise noninvasive cells. 7,8 Inhibition of invasion in the presence of ET-18-OMe was explained by induction of host tissue resistance, 9 but the antithetic effects on cell-cell adhesion and the invasion-promoting effect of ET-18-OMe have not been explained.Adhesion and invasion are the result of a balance between promoter and suppressor molecules modulated by multiple intraand extracellular factors. The presence of the anti-adhesion molecule episialin on the 2 types of MCF-7 cells was observed in a previous study. 2 The aim of our study was to see whether ET-18-OMe affects invasion and adhesion through modulation of episialin and E-cadherin.Episialin is a heterodimeric molecule composed of a membrane anchor and an extracellular moiety. The membrane anchor consists of a cytoplasmatic domain of 68 amino acids and a transmembrane domain. The extracellular moiety consists of a large mucin domain containing a large number of O-linked glycans. 10 The carbohydrate side chains carry many sialic acid residues conveying a highly negative charge upon episialin. 11 Episialin is located at the apical surface of most glandular epithelial cells; it is over-expressed in carcinomas, often distributed over the entire cell surface. 11 Overexpression of episialin inhibits integrin-mediated cell adhesion to

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“…Cells were fixed with 4% paraformaldehyde for 10 min and washed four times in 10 mM Tris-HCl, 1 mM CaCl 2 , 1mM MgCl 2 , pH 7.4 (TBS). Cells were blocked with 2% periodate-treated [26] bovine serum albumin (BSA) in TBS for 30 min. The cells were washed four times in TBS and then incubated with fluorescein isothiocyanate (FITC)-labelled lectins (Table 1) for 1 h in the dark at the following concentrations: SNA-I, MAA, PNA and ECA at 20 μg/mL and WFA at 10 μg/ml in TBS.…”

Section: Lectin Cytochemistrymentioning

confidence: 99%

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

Kilcoyne

Sharma

McDevitt

et al. 2012

Biochemical and Biophysical Research Communications

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Glycosylation is found ubiquitously throughout the central nervous system (CNS).Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration.Several CSPGs are upregulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA).Usually α-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining, and by day 8 in the case of WFA. This study demonstrated neuronal cell surface glycosylation changes in an inhibitory environment and indicated a return to normal glycosylation after treatment with ChABC, which may be promising for identifying potential therapies for neuronal regeneration strategies.

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Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets

Cited by 231 publications

References 10 publications

Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

Alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl- glycerophosphocholine induces invasion through episialin-mediated neutralization of E-cadherin in human mammary MCF-7 cellsin vitro

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

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