Search results for "immunoblotting"

Cross-resistance and mechanism of resistance to Cry1Ab toxin from Bacillus thuringiensis in a field-derived strain of European corn borer, Ostrinia n…

2011

The cross-resistance spectrum and biochemical mechanism of resistance to the Bacillus thuringiensis Cry1Ab toxin was studied in a field-derived strain of Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae) that was further selected in the laboratory for high levels (>1000-fold) of resistance to Cry1Ab. The resistant strain exhibited high levels of cross-resistance to Cry1Ac and Cry1Aa but only low levels of cross-resistance (<4-fold) to Cry1F. In addition, there was no significant difference between the levels of resistance to full-length and trypsin-activated Cry1Ab protein. No differences in activity of luminal gut proteases or altered proteolytic processing of the toxin were observed in…

Mapping the protein composition oftrans-Golgi network (TGN)-derived carrier vesicles from polarized MDCK cells

1998

In polarized MDCK cells, proteins and lipids are sorted in the trans-Golgi network /TGN) and packaged into different vesicular carriers that are delivered to the apical or basolateral cell surface. To gain insight into the sorting and trafficking machinery, we have previously isolated TGN-derived carrier vesicles from perforated MDCK cells. The composition of immuno-isolated apical and basolateral carriers was mapped by two-dimensional (2-D) gel electrophoresis. Here we describe the identification of several components of the vesicle fraction by using three different methods. 2-D gel comigration was performed with carrier vesicles isolated from metabolically labeled MDCK cells and human epi…

Proteomic analysis of the photosystem I light-harvesting antenna in tomato (Lycopersicon esculentum).

2004

Until now, more genes of the light-harvesting antenna of higher-plant photosystem I (PSI) than proteins have been described. To improve our understanding of the composition of light-harvesting complex I (LHCI) of tomato (Lycopersicon esculentum), we combined one- and two-dimensional (1-D and 2-D, respectively) gel electrophoresis with immunoblotting and tandem mass spectrometry (MS/ MS). Separation of PSI with high-resolution 1-D gels allowed separation of five bands attributed to proteins of LHCI. Immunoblotting with monospecific antibodies and MS/MS analysis enabled the correct assignment of the four prominent bands to light-harvesting proteins Lhcal -4. The fifth band was recognized by o…

New perspectives on the renal slit diaphragm protein podocin.

2011

Podocin is a critical component of the glomerular filtration barrier, its mutations causing recessive steroid-resistant nephrotic syndrome. A GenBank analysis of the human podocin (NPHS2) gene resulted in the possible existence of a new splice variant of podocin in the kidney, missing the in-frame of exon 5, encoding the prohibitin homology domain. Using RT–polymerase chain reaction and immunoblotting followed by sequence analysis, we are for the first time able to prove the expression of a novel podocin isoform (isoform 2), exclusively and constitutively expressed in human podocytes. Furthermore, we reveal singular extrarenal podocin expression in human and murine testis. Our data show the…

Stress-70 proteins in marine mussel Mytilus galloprovincialis as biomarkers of environmental pollution: a field study.

2004

In the present work we have investigated levels of stress-70 proteins in the gills of mussel Mytilus galloprovincialis collected seasonally from subtidal rocky shores at 6 different sites of the Rovinj coastal area (Northern Adriatic, Croatia). 1-D analysis (SDS-PAGE) using monoclonal mouse antibodies anti-HSP70 detected two bands of stress-70 proteins, 70 and 72 kDa constitutively present during the year. 2-D analysis (IEF+SDS-PAGE) proved that the antibodies used detected HSP70 (pI 5.7–5.9) and HSP72 (pI 5.5–5.6). The quantification of stress-70 proteins was possible using 200 ng of external HSP70 protein standard included on every blot. Maximal levels of HSP72 and HSP70 were observed in …

Biological standards for hepatitis B virus assays.

1992

Anti-rat liver microsomal and cytosolic antibodies in hepatitis C virus infection.

1994

In order to assess the frequency of autoimmunity markers in hepatitis C virus infection, 229 RIBA 2 HCV positive individuals were tested by ELISA and Immunoblot assay using as antigen rat liver microsomal and cytosolic proteins. Twenty-one out of 229 individuals (9%) showed anti-rat liver microsome antibodies by ELISA, but the titre was low (1:100 to 1:1,600). In Immunoblot, only 5 of these 21 ELISA positive sera recognized also rat liver microsomal proteins (MW between 30 to 64 kDa). Antibodies against rat liver cytosolic proteins were found by ELISA in 14 out of 229 individuals (6%). Three of them showed a reactivity in Immunoblot to 42 kDa or 55 kDA proteins. In conclusion, HCV infection…

Cellular responses and HSP70 expression during wound healing in Holothuria tubulosa (Gmelin, 1788).

2014

Wound repair is a key event in the regeneration mechanisms of echinoderms. We studied, at the behavioural, cellular and molecular levels, the wound healing processes in Holothuria tubulosa after injuries to the body wall. The experiments were performed for periods of up to 72 h, and various coelomocyte counts, as well as the expression of heat shock proteins (HS27, HSP70 and HSP90), were recorded. Dermal wound healing was nearly complete within 72 h. In the early stages, we observed the injured animals twisting their bodies to keep their injuries on the surface of the water for the extrusion of the buccal pedicles. At the cellular level, we found time-dependent variations in the circulating…

Sodium Solute Symporter and Cadherin Proteins Act as Bacillus thuringiensis Cry3Ba Toxin Functional Receptors in Tribolium castaneum*

2013

Understanding how Bacillus thuringiensis (Bt) toxins interact with proteins in the midgut of susceptible coleopteran insects is crucial to fully explain the molecular bases of Bt specificity and insecticidal action. In this work, aminopeptidase N (TcAPN-I), E-cadherin (TcCad1), and sodium solute symporter (TcSSS) have been identified by ligand blot as putative Cry3Ba toxin-binding proteins in Tribolium castaneum (Tc) larvae. RNA interference knockdown of TcCad1 or TcSSS proteins resulted in decreased susceptibility to Cry3Ba toxin, demonstrating the Cry toxin receptor functionality for these proteins. In contrast, TcAPN-I silencing had no effect on Cry3Ba larval toxicity, suggesting that th…

Complex Cellular Responses of Helicobacter pylori-Colonized Gastric Adenocarcinoma Cells ▿

2011

ABSTRACT Helicobacter pylori is an important class I carcinogen that persistently infects the human gastric mucosa to induce gastritis, gastric ulceration, and gastric cancer. H. pylori pathogenesis strongly depends on pathogenic factors, such as VacA (vacuolating cytotoxin A) or a specialized type IV secretion system (T4SS), which injects the oncoprotein CagA (cytotoxin-associated gene A product) into the host cell. Since access to primary gastric epithelial cells is limited, many studies on the complex cellular and molecular mechanisms of H. pylori were performed in immortalized epithelial cells originating from individual human adenocarcinomas. The aim of our study was a comparative anal…