Sodium dodecyl sulfate agarose gel electropheresis and electroelution of high molecular weight human salivary mucin

Gabriel, Matthias; Zentner, Andrej

doi:10.1007/s00784-005-0007-2

Cited by 8 publications

(6 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To avoid this problem, we repeated the experiment, this time including SDS (0.2%) in the agarose gels. Our rationale was that SDS causes denaturated polypeptides to adopt a highly extended conformation ( Gabriel and Zentner, 2005; Shinagawa et al., 1979 ), which might alter the electrophoretic properties of protein- but not DNA-mediated catenanes. Analysis of samples by SDS-PAGE and western blotting confirmed that bBBr induced efficient crosslinking of the various cysteine pairs; note that the cohesin proteins detected in the gradient fractions are not exclusively or even mainly those associated with minichromosome DNA ( Figure 4 B).…”

Section: Resultsmentioning

confidence: 99%

Cohesin's Concatenation of Sister DNAs Maintains Their Intertwining

et al. 2011

View full text Add to dashboard Cite

SummaryThe contribution of DNA catenation to sister chromatid cohesion is unclear partly because it has never been observed directly within mitotic chromosomes. Differential sedimentation-velocity and gel electrophoresis reveal that sisters of 26 kb circular minichromosomes are held together by catenation as well as by cohesin. The finding that chemical crosslinking of cohesin's three subunit interfaces entraps sister DNAs of circular but not linear minichromosomes implies that cohesin functions using a topological principle. Importantly, cohesin holds both catenated and uncatenated DNAs together in this manner. In the vicinity of centromeres, catenanes are resolved by spindle forces, but linkages mediated directly by cohesin resist these forces even after complete decatenation. Crucially, persistence of catenation after S phase depends on cohesin. We conclude that by retarding Topo II-driven decatenation, cohesin mediates sister chromatid cohesion by an indirect mechanism as well as one involving entrapment of sister DNAs inside its tripartite ring.

show abstract

Section: Resultsmentioning

confidence: 99%

Cohesin's Concatenation of Sister DNAs Maintains Their Intertwining

et al. 2011

View full text Add to dashboard Cite

show abstract

“…SDS-Agarose gel electrophoresis was used to analyze mucin expression as described previously [29]. Cells expressing miR-200c or a scrambled control were harvested and protein was extracted using NP-40 cell lysis buffer (150mM NaCl, 50mM Tris-HCl, 1% NP-40, pH8.0) supplemented with protease inhibitors (Promega, Madison, WI, USA).…”

Section: Methodsmentioning

confidence: 99%

MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

et al. 2013

View full text Add to dashboard Cite

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.

show abstract

“…SDS-agarose gel electrophoresis for mucin and mucin glycans was carried out as described previously. 27 Briefly, cell lysates (50-100 lg proteins) were resolved on SDS (0.1%)-agarose (2%) gel electrophoresis and transferred overnight to PVDF membrane by capillary transfer. Membranes were blocked in 5% milk in TBS-T (Tris-buffered saline and 0.1% Tween 20, pH 7.4) and incubated with the following primary antibodies, 5E5 (mouse IgG against S/Tn epitope on MUC1) a kind gift from Dr. Henrik Clausen, Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark, and mouse anti-b-tubulin IgG (Developmental Studies Hybridoma Bank, Iowa).…”

Section: Sds-agarose Electrophoresismentioning

confidence: 99%

Expression of core 3 synthase in human pancreatic cancer cells suppresses tumor growth and metastasis

Radhakrishnan

Grandgenett

Mohr

et al. 2013

Intl Journal of Cancer

View full text Add to dashboard Cite

Core 3 derived glycans, a major type of O-glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy, because of loss of expression of functional β3-N-acetylglucosaminyltransferase-6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re-expressing core 3 synthase in pancreatic cancer cells that had lost expression. We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. We therefore re-expressed in human pancreatic cancer cells (Capan-2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared with vector control cells. Expression of core 3 O-glycans induced altered expression of β1 integrin, decreased activation of focal adhesion kinase, led to the down regulation of expression of several genes including REG1α and FGFR3, and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. These findings indicate that expression of core 3 derived O-glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins.

show abstract

Sodium dodecyl sulfate agarose gel electropheresis and electroelution of high molecular weight human salivary mucin

Cited by 8 publications

References 12 publications

Cohesin's Concatenation of Sister DNAs Maintains Their Intertwining

Cohesin's Concatenation of Sister DNAs Maintains Their Intertwining

MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

Expression of core 3 synthase in human pancreatic cancer cells suppresses tumor growth and metastasis

Contact Info

Product

Resources

About