protease inhibitors  (Boehringer Mannheim)

Journal: Journal of Virology

Article Title: Antiviral Activity of the Proteasome on Incoming Human Immunodeficiency Virus Type 1

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Figure Lengend Snippet: Analysis of the effect of various protease inhibitors on HIV infection and on LTR activity. (A) Effect of MG132 on HIV-1 LTR activity. P4 cells were transfected with 1 μg of pLTRX-Luc and the indicated amounts of pCMV-Tat DNA. After 24 h, cells were pulse-incubated for 1 h with or without MG132 (50 μM), and 5 h later, luciferase activities in cell extracts were measured. Results are expressed as RLU per microgram of cellular protein. Values are means of triplicate determinations, and variation between each point was below 10%. (B) P4 cells were infected with HIV-1 (left panel) or HIVΔenv(VSV) (right panel) in the presence of the proteasome inhibitors MG132 (50 μM) or lactacystin (40 μM) or with calpain inhibitor I (50 μM) or calpain inhibitor II (50 μM) for 1 h and washed. Infections were revealed 24 h later by measuring β-galactosidase activity (optical density [O.D.] units). Data are representative of three independent experiments.

Article Snippet: Lactacystin was obtained from BioMol, and calpain inhibitors I and II were obtained from Boehringer Mannheim.

Techniques: Infection, Activity Assay, Transfection, Incubation, Luciferase

Journal: The Journal of Cell Biology

Article Title: Activation of ?V?3 on Vascular Cells Controls Recognition of Prothrombin

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Figure Lengend Snippet: Effects of protein kinase C and calpain inhibitors ( A ) and cytochalasin B ( B and C ) on adhesion of PMA- and MnCl 2 -treated HUVEC to immobilized prothrombin. HUVEC adhesion was measured (see legend to Fig. 3 and Materials and Methods) in the absence ( open bar ) or presence of 200 nM PMA ( black bars ) or in the presence of 0.5 mM MnCl 2 ( gray bars ). In A , cells were pretreated with calpeptin (50 μg/ml), calpain inhibitor I and II (100 μg/ml each), bisindolylmaleimide V ( BIM V ) and bisindolylmaleimide I ( BIM I ; 20 nM each) or calphostin C light-activated; 1 μM). In B , adherent cells in the absence or presence (0.1 μm) of cytochalasin B were photographed at 40×. Bar, 50 μm. In C , adhesion of stimulated cells was measured after 50 min in the absence or presence of cytochalasin B. Adhesion in the presence of 200 nM PMA without inhibitors was assigned a value of 100%. The data shown are means and SD from three experiments.

Article Snippet: Calpain inhibitors I and II were from Boehringer Mannheim (Mannheim, Germany).

Techniques:

Journal: Genes & Development

Article Title: BCoR, a novel corepressor involved in BCL-6 repression

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Figure Lengend Snippet: Full-length BCL-6 and BCoR interact in vitro and in vivo, the POZ domain of BCL-6 is both necessary and sufficient for interaction with BCoR in vitro, and BCL-6 and BCoR colocalize in the nucleus. ( A ) Structure of the proteins used in B and C . (Solid oval) The POZ domain; (solid rectangle) zinc fingers. (*) The myc epitope tag. ( B ) Coimmunoprecipitation of BCL-6 and BCoR in vitro. Pair-wise combinations of [ 35 S]methionine-labeled nontagged BCL-6 derivatives and myc-tagged BCoR were produced by cotranslation and immunoprecipitated with the α-myc antibody. The immune complexes were recovered and analyzed by SDS-PAGE (10% for lanes 1–5; 17% for lanes 6,7 ). The intensity of myc–BCoR in lanes 6 and 7 appears stronger due to compression on the 17% gel of non-full-length myc-BCoR proteins. Input represents 20% of the total used. (●) The expected migration of the nontagged BCL-6 derivatives. ( C ) Coimmunoprecipitation of BCL-6 and BCoR in vivo. 293 cells were transfected with 3 μg of BCL-6 expression plasmid alone (lane 2 ) or cotransfected with 3 μg each of BCL-6 and myc-tagged BCoR expression plasmids (lanes 1,3 ). Cell lysates were immunoprecipitated with α-myc antibody (lanes 2,3 ) and the recovered proteins were detected by Western blot analysis using N-3 α-BCL-6 and α-myc antibodies. The input (lane 1 ) corresponds to 1/50 of the lysate used in the immunoprecipitation (lane 3 ). ( D ) Subcellular localization of BCoR and BCoR-S. Immunofluorescence was performed on HeLa cells transfected with 1.2 μg of either myc-tagged BCoR or BCoR-S expression plasmids. Multiple Z series confocal microscopy images of the given fields were compiled for each image of BCoR and BCoR-S. ( E ) Colocalization of BCL-6 and BCoR. Coimmunofluorescence was performed on HeLa cells cotransfected with 0.6 μg each of BCL-6 and myc-tagged BCoR, and the data collected as in D . The compiled images of BCoR and BCL-6 were merged to detect colocalization.

Article Snippet: Transfected cells were washed twice with PBS and lysed in 500 μl of cold immunoprecipitation (IP) buffer (PBS, 10% glycerol, 0.5% NP-40, and a complete protease inhibitor cocktail from Boehringer-Mannheim) for 10 min on ice.

Techniques: In Vitro, In Vivo, Zinc-Fingers, Labeling, Produced, Immunoprecipitation, SDS Page, Migration, Transfection, Expressing, Plasmid Preparation, Western Blot, Immunofluorescence, Confocal Microscopy

Journal: Molecular and Cellular Biology

Article Title: Role of the I?B Kinase Complex in Oncogenic Ras- and Raf-Mediated Transformation of Rat Liver Epithelial Cells

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Figure Lengend Snippet: IκB-α protein is more phosphorylated and has a shorter half-life in F22-Ras and TH-Raf cells than in wt RLEs. (A) Phosphorylation state. Cytoplasmic extracts (40 μg) from exponentially growing wt, F22-Ras and TH-Raf RLEs, treated for 1 h with 40 μM calpain inhibitor I to inhibit IκB-α degradation, were resolved on isoelectric focusing gels, separated according to molecular weight by SDS-PAGE, and subjected to immunoblot analysis using an antibody preparation raised against the IκB-α product (SC-371). (B) Half-life of decay. Exponentially growing RLEs were treated with the protein synthesis inhibitor emetine (emet; 10 μg/ml) for 1 to 4 h. Cytoplasmic extracts (20 μg) were then subjected to immunoblot analysis for IκB-α as described above.

Article Snippet: Where indicated, cells were incubated for 1 h with 40 μM calpain inhibitor I (Boehringer Mannheim, Indianapolis, Ind.).

Techniques: Molecular Weight, SDS Page