Protein blotting: Principles and applications

Gershoni, Jonathan M.; Palade, George E.

doi:10.1016/0003-2697(83)90128-8

Cited by 835 publications

(242 citation statements)

References 88 publications

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“…Proteins separated with SDS -PAGE were transferred to nitrocellulose membranes; the transfers were performed overnight at 30 V in a cooled (41C) reservoir containing 25 mM Tris (tris (hydroxymethyl) aminomethane), 192 mM glycine, and 20% methanol (pH 8.3) (Towbin et al, 1979) transfer buffer. The nitrocellulose membranes were then removed from the blot apparatus and placed in a solution of Ponceau S stain (0.5% Ponceau S and 1% glacial acetic acid in water) to verify equal loading of protein in the control and treated samples (Gershoni and Palade, 1983).…”

Section: Western Immunoblottingmentioning

confidence: 99%

Atiprimod blocks STAT3 phosphorylation and induces apoptosis in multiple myeloma cells

et al. 2005

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Multiple myeloma (MM) accounts for 1 % of all cancer deaths. Although treated aggressively, almost all myelomas eventually recur and become resistant to treatment. Atiprimod 5] decane dimaleate) has exerted anti-inflammatory activities and inhibited oeteoclast-induced bone resorption in animal models and been well tolerated in patients with rheumatoid arthritis in phase I clinical trials. Therefore, we investigated its activity in MM cells and its mechanism of action. We found that Atiprimod inhibited proliferation of the myeloma cell lines U266-B1, OCI-MY5, MM-1, and MM-1R in a timeand dose-dependent manner. Atiprimod blocked U266-B1 myeloma cells in the G 0 /G 1 phase, preventing cell cycle progression. Furthermore, Atiprimod inhibited signal transducer and activator of transcription (STAT) 3 activation, blocking the signalling pathway of interleukin-6, which contributes to myeloma cell proliferation and survival, and downregulated the antiapoptotic proteins Bcl-2, Bcl-X L , and Mcl-1. Incubation of U266-B1 myeloma cells with Atiprimod induced apoptosis through the activation of caspase 3 and subsequent cleavage of the DNA repair enzyme poly(adenosine diphosphate-ribose) polymerase. Finally, Atiprimod suppressed myeloma colony-forming cell proliferation in fresh marrow cells from five patients with newly diagnosed MM in a dose-dependent fashion. These data suggest that Atiprimod has a role in future therapies for MM.

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Section: Western Immunoblottingmentioning

confidence: 99%

Atiprimod blocks STAT3 phosphorylation and induces apoptosis in multiple myeloma cells

et al. 2005

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“…After electrophoresis, the gel was blotted to polyvinylidene difluoride membrane and stained with Amido Black (17). The membrane was sent to the Protein Microsequencing Facility of the Wistar Institute (Philadelphia, PA) for protease digestion and amino acid sequencing.…”

Section: Materials-cholesterylmentioning

confidence: 99%

Purification and Characterization of a Neutral, Bile Salt-independent Retinyl Ester Hydrolase from Rat Liver Microsomes

Sun¹,

Alexson²,

Harrison³

1997

Journal of Biological Chemistry

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A neutral, bile salt-independent retinyl ester hydrolase (NREH) has been purified from a rat liver microsomal fraction. The purification procedure involved detergent extraction, DEAE-Sepharose ion exchange, PhenylSepharose hydrophobic interaction, Sephadex G-100 and Sephacryl S-200 gel filtration chromatographies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated enzyme has an apparent molecular mass of approximately 66 kDa under denaturing conditions on SDS-PAGE. Analysis of the amino acid sequences of four peptides isolated after proteolytic digestion revealed that the enzyme is highly homologous with other rat liver carboxylesterases. In particular, the sequences of the four peptides of the NREH (60 amino acids total) were identical to those of a rat carboxylesterase expressed in the liver ( Hydrolysis of retinyl esters occurs during both the hepatic uptake of newly absorbed dietary vitamin A and during the mobilization of retinyl ester stores from the liver. Thus, hepatic enzymes catalyzing the hydrolysis of retinyl esters are important in the body's overall vitamin A homeostasis. Earlier studies focused on a neutral, bile salt-dependent retinyl ester hydrolase that is now understood to be the enzyme carboxylester lipase (see Ref. 1 for a review). This enzyme is secreted by the pancreas into the intestinal lumen, where it can presumably hydrolyze dietary retinyl esters and other lipid esters (2-5). The enzyme is also secreted by the liver (6), and its role in hepatic retinyl ester metabolism is under investigation but is presently unclear. Carboxylester lipase has been cloned from several species, including the rat (see Ref. 7 for a review). In addition to carboxylester lipase, a number of tissues contain bile salt-independent retinyl ester hydrolase activities that have as yet not been fully purified or characterized biochemically (4, 8 -10). For example, we have previously demonstrated the occurrence of both acid and neutral retinyl ester hydrolase activities associated with rat liver microsomal preparations enriched in plasma membranes and endosomes (4, 10). We recently reported studies demonstrating the co-localization of newly delivered chylomicron retinyl esters and bile salt-independent retinyl ester hydrolase enzyme activities in the same plasma membrane/endosome fractions, and the in vitro hydrolysis of chylomicron remnant retinyl esters by these fractions (11). These studies suggested a probable role for these enzymes in the initial hepatic metabolism of chylomicron retinyl esters.In the present investigations, we have undertaken the purification and characterization of a neutral, bile salt-independent retinyl ester hydrolase from rat liver microsomes. The isolated enzyme has an apparent molecular mass of 66 kDa and catalyzes the hydrolysis of retinyl esters at higher rates than triglyceride hydrolysis. It shows no cholesteryl ester hydrolase activity. Partial amino acid sequence analysis and immunoblot analysis show that the enzyme is highly related to or ident...

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“…The fragments were separated with PAGE [19]. After electrophoresis the gels were incubated in 25 mM Tris and 20% methanol (pH 10.4)for 15min, and the proteins were transferred to Novilon" membranes (Millipore Corp., Bedford, Mass) by using a Multiphor II Novablot" system (LKB, Bromma, Sweden) at 150 rnA for 90 min [20]. The membrane was then slowly shaken in a blocking buffer (10 mM Tris, 1 mM EDTA, 133 mMNaCI, 10010 skim milk, 0.05% Triton X-l00, and 2% sodium azide) at room temperature (1'\.123 C) for 2 h. Subsequently the membrane was incubated in skim milk (Difco, Detroit) containing an antibody to fibronectin raised in rabbits (Behring, Marburg, Federal Republic of Germany) at a dilution of 1:4000 followed by three washing steps in PBS (pH 7.4), Ca" and Mg" free.…”

Section: Fibronectin Cleavage In Cystic Fibrosismentioning

confidence: 99%

Fibronectin-Cleaving Activity in Bronchial Secretions of Patients with Cystic Fibrosis

Suter

Schaad²,

Morgenthaler³

et al. 1988

Journal of Infectious Diseases

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In cystic fibrosis, colonization of the airways with Pseudomonas aeruginosa follows colonization with Staphylococcus aureus and is related to accelerated deterioration of pulmonary function. Because P. aeruginosa adheres better to cell surfaces devoid of fibronectin, we searched for fibronectin-cleaving activity in bronchial secretions and saliva from 24 patients with cystic fibrosis who were followed up for 4.5 y and from two control groups. Proteolytic activity against 12sI-labeled fibronectin was continuously present in cysticfibrosis bronchial secretions; significantly higher fibronectin-cleaving activity was found in older vs. younger patients, in patients with advanced disease stages determined by a five-stage scoring system, and in those colonized with P. aeruginosa. The fibronectin-cleaving activity was due to neutrophil elastase and cathepsin G. Cystic fibrosis bronchial secretions had proteolytic activity against surface fibronectin of airway mucosal cells. Thus fibronectin-cleaving activity of bronchial secretions rather than of saliva may favor P. aeruginosa colonization of the upper respiratory tract in individuals with cystic fibrosis.Most individuals with cystic fibrosis who survive beyond the neonatal period die from respiratory failure, which is the consequence of chronic, progressively destructive bronchitis [1][2][3]. Clinical and pathological observations show extensive tissue destruction in the airways, which first causes obstruction and obliteration of small airways and thereafter destruction of the walls of large airways [4,5]. Early in the disease course the bacterial pathogens frequently found in the bronchial secretions are Staphylococcus aureus and Haemophilus influenzae. Later the airways of these subjects are invariably colonized with Pseudomonas aeruginosa, an event that initiates an acclerated deterioration of lung functions [3] as wellas an excessive systemic immune response [6]. These pathogens cannot be eliminated from the airways by antimicrobial therapy, and because the deleterious effect of P. aeruginosa infection in individuals with cystic fibrosis is well established, the identification of factors favoring colonization of the airways with P. aeruginosa is important. Coloniza-

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Protein blotting: Principles and applications

Cited by 835 publications

References 88 publications

Atiprimod blocks STAT3 phosphorylation and induces apoptosis in multiple myeloma cells

Atiprimod blocks STAT3 phosphorylation and induces apoptosis in multiple myeloma cells

Purification and Characterization of a Neutral, Bile Salt-independent Retinyl Ester Hydrolase from Rat Liver Microsomes

Fibronectin-Cleaving Activity in Bronchial Secretions of Patients with Cystic Fibrosis

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