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Boehringer Mannheim nonidet p 40
Nonidet P 40, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Lysis:

Article Title: Obesity-Mediated Autophagy Insufficiency Exacerbates Proteinuria-induced Tubulointerstitial Lesions
Article Snippet: .. Renal cortices and cultured PTECs were homogenized in an ice-cold lysis buffer containing 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.0), 0.1% SDS, 1% Nonidet P-40, and protease inhibitor cocktail (Boehringer Mannheim, Lewes, UK). .. The samples were resolved by 10% or 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon, Bedford, MA).

Article Title: Human and mouse homologs of Schizosaccharomyces pombe rad1+ and Saccharomyces cerevisiae RAD17: linkage to checkpoint control and mammalian meiosis
Article Snippet: .. To prepare U2OS and HeLa whole cell extract, cells were lysed in 300 μl of lysis buffer I [50 m m Tris (pH 8), 150 m m NaCl, 5 m m EDTA, 0.5% Nonidet P-40, and complete protease inhibitors; Boehringer Mannheim]. .. The lysis mixture was incubated on ice for 20 min and cleared by centrifugation at 12,000 g for 10 min at 4°C.

Article Title: Strain Differences in Behavioral and Cellular Responses to Perinatal Hypoxia and Relationships to Neural Stem Cell Survival and Self-Renewal
Article Snippet: .. NSCs were homogenized in lysis buffer composed of 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% Nonidet P-40, 10% glycerol, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Boehringer Mannheim GmbH, Mannheim, Germany). ..

Article Title: Evidence of a Role for Nonmuscle Myosin II in Herpes Simplex Virus Type 1 Egress
Article Snippet: .. To that end, HeLa cells (approximately 2 × 1010 ) were suspended in 100 ml of lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5 mM (PMSF), and Complete protease inhibitor cocktail according to the instructions of the manufacturer (Boehringer Mannheim). .. After incubation for 45 min on ice with occasional mixing, the lysate was diluted 1:1 in buffer containing 50 mM Tris (pH 7.0), 50 mM NaCl, and 0.5 mM PMSF.

Incubation:

Article Title: Cowpox virus encodes a fifth member of the tumor necrosis factor receptor family: A soluble, secreted CD30 homologue
Article Snippet: .. Portions of samples were resuspended in 90 mM sodium phosphate buffer, pH 7.5, containing 1% SDS and 1% 2-mercaptoethanol, boiled for 5 min, diluted with a 200 mM sodium phosphate buffer containing 1.0% Nonidet P-40, and incubated 16 hr at 37°C, with or without 0.5 unit of N-glycosidase F (Boehringer Mannheim). .. Proteins from the equivalent of 0.5 × 108 cells were resolved by SDS/PAGE in 10% polyacrylamide gels, and transferred to Immobilon-P membranes (Millipore).

other:

Article Title: Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation
Article Snippet: Nonidet P-40 and proteinase K were purchased from Boehringer Mannheim (Mannheim, Germany).

Cell Culture:

Article Title: Obesity-Mediated Autophagy Insufficiency Exacerbates Proteinuria-induced Tubulointerstitial Lesions
Article Snippet: .. Renal cortices and cultured PTECs were homogenized in an ice-cold lysis buffer containing 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.0), 0.1% SDS, 1% Nonidet P-40, and protease inhibitor cocktail (Boehringer Mannheim, Lewes, UK). .. The samples were resolved by 10% or 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon, Bedford, MA).

Protease Inhibitor:

Article Title: Obesity-Mediated Autophagy Insufficiency Exacerbates Proteinuria-induced Tubulointerstitial Lesions
Article Snippet: .. Renal cortices and cultured PTECs were homogenized in an ice-cold lysis buffer containing 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.0), 0.1% SDS, 1% Nonidet P-40, and protease inhibitor cocktail (Boehringer Mannheim, Lewes, UK). .. The samples were resolved by 10% or 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon, Bedford, MA).

Article Title: Strain Differences in Behavioral and Cellular Responses to Perinatal Hypoxia and Relationships to Neural Stem Cell Survival and Self-Renewal
Article Snippet: .. NSCs were homogenized in lysis buffer composed of 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% Nonidet P-40, 10% glycerol, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Boehringer Mannheim GmbH, Mannheim, Germany). ..

Article Title: Evidence of a Role for Nonmuscle Myosin II in Herpes Simplex Virus Type 1 Egress
Article Snippet: .. To that end, HeLa cells (approximately 2 × 1010 ) were suspended in 100 ml of lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5 mM (PMSF), and Complete protease inhibitor cocktail according to the instructions of the manufacturer (Boehringer Mannheim). .. After incubation for 45 min on ice with occasional mixing, the lysate was diluted 1:1 in buffer containing 50 mM Tris (pH 7.0), 50 mM NaCl, and 0.5 mM PMSF.

Article Title: A novel regulatory role of glucose transporter of Escherichia coli: membrane sequestration of a global repressor Mlc
Article Snippet: .. The cell pellets were suspended in 0.5 ml of ice-cold 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, CompleteTM Protease inhibitor Cocktail Tablets (Boehringer Mannheim). .. The cells were sonicated for 5 s four times at 0°C, with 10 s rest periods in between, using a special micro tip of a Heat Systems Ultrasonics Sonicator.

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  • 80
    Boehringer Mannheim precondensed triton x 114
    Recombinant and naturally processed sFasLs show marginal apoptotic activity on human cells. ( A ) Processing of membrane-bound FasL to  sFasL. Cellular extracts of a 293 cell clone stably transfected with human FasL cDNA, were separated into Triton X-114–insoluble ( Insoluble ) and –soluble  ( Soluble ) fractions. Cell supernatants ( Medium ) were separated into detergent and aqueous fractions by Triton X-114 phase separation. Samples were analyzed by Western blotting for the presence of FasL using an antibody specific for the extracellular segment of the ligand. The indicated FasL and sFasL  bands were absent in mock transfected controls ( D , and data not shown). The processed sFasL was purified by Fas/Fc affinity chromatography and visualized by Coomassie blue staining ( CB ). ( B ) 293 cells were transfected with mouse FasL cDNA. Total, unfractionated cellular extracts and cell supernatants  were analyzed by Western blotting as described for  A . ( C ) Site of proteinase-induced cleavage of FasL. Metalloproteinase(s) present in 293 cells cleave(s)  membrane-bound FasL at the Ser126–Leu127 bond, liberating a soluble form of FasL. The cleavage site of the TNF-α precursor is shown for comparison. ( D ) The effect of cleavage site mutations on FasL processing. Wild-type ( WT ) FasL as well as FasL mutated at the P1 site ( S126E ), at the P1′ site  ( L127E ), and at both the P1 and P1′ sites ( S126E/L127E ) were transiently transfected into 293T cells. Insoluble proteins (containing membrane-associated FasL) and medium (containing processed sFasL) were analyzed by Western blotting as described above. To achieve similar loading of soluble and  cell-associated FasL, the supernatants corresponding to 10 times more cells were loaded on the gel. A nonspecific band at 60 kD indicates equal loading of  the gel. In the control lane, cell extracts of nontransfected 293 cells were analyzed. ( E ) Activity of naturally processed ( open squares ) and recombinant  ( closed squares ) sFasL on Jurkat T cells.
    Precondensed Triton X 114, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Boehringer Mannheim immunoprecipitation assay buffer
    Molecular characterization of the calsenilin knock-out mouse. A , Gene targeting strategy. Top, Wild-type calsenilin locus showing exon 2 and the position of the 5′ and 3′ probes used for genotyping. Bottom, Mutant locus in which exon 2 has been replaced with an IRES, the marker gene β -gal , and the neomycin (neo) resistance gene, the latter under the control of the MC1 promoter. B , Southern blot analysis of genomic DNA from calsenilin +/+, +/-, and -/- mice. Left, Hind III digest; right, Nco I digest. C , Northern blot analysis of mRNA extracted from the brains of calsenilin +/+, +/-, and -/- mice. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is included as a loading control. D ). The asterisk indicates a band migrating just above mouse calsenilin, and the position of IgG (from the <t>immunoprecipitation)</t> is also indicated.
    Immunoprecipitation Assay Buffer, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Boehringer Mannheim endo f incubation buffer
    (A) Immunoblot analysis of NS1 and its derivatives from primary or persistent JEV infections. Cell lysates from primary infection of BHK-21 cells (lanes 1 and 4) and from persistent infection of B2-5/RP9 (lanes 2 and 5) and C2-2 cells (lanes 3 and 6) were separated by SDS–10% PAGE and immunoblotted with anti-NS1 monoclonal antibody JE7/45-2. Protein samples were treated either with boiling (lanes 4 to 6) or without boiling (lanes 1 to 3). The wild-type NS1 proteins are indicated by arrows. The truncated NS1 and NS1′ are marked by asterisks. Molecular mass markers (kilodaltons) are on the left of the figure. (B) Glycosylation analysis of NS1 proteins by <t>endo-F</t> digestion. Cell lysates from JEV primary infection of BHK-21 (lanes 1 and 4) and from persistent infection of B2-5/RP9 (lanes 2 and 5) and C2-2 cells (lanes 3 and 6) were immunoprecipitated by anti-NS1 monoclonal antibodies and then were treated either without (lanes 1 to 3) or with (lanes 4 to 6) endo-F at <t>37°C</t> for 16 h. The deglycosylated NS1 and NS1′, as well as their truncated proteins p45 and p39 (lanes 4 to 6), are indicated by open triangles; their counterparts without endo-F digestion (lanes 1 to 3) are marked by arrows. (C) Localization of the truncation region to the N termini of NS1 and NS1′ proteins derived from the persistently JEV-infected cells by immunoprecipitation with anti-NS1 monoclonal antibodies. 35 S-labeled lysates from primarily infected BHK-21 (lanes 1 and 4) and persistently infected B2-1/RP9 (lanes 2 and 5) and B2-5/RP9 (lanes 3 and 6) cells were precipitated by anti-NS1 D2/39.1 (lanes 1 to 3) or JE7/45-2 (lanes 4 to 6) and then separated by SDS–10% PAGE. The wild-type NS1 and NS1′ are marked with arrows, and the truncated protein p39 is marked by an asterisk. Note that only antibody JE7/45-2 recognized the truncated NS1 (lanes 5 and 6).
    Endo F Incubation Buffer, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 80/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Recombinant and naturally processed sFasLs show marginal apoptotic activity on human cells. ( A ) Processing of membrane-bound FasL to  sFasL. Cellular extracts of a 293 cell clone stably transfected with human FasL cDNA, were separated into Triton X-114–insoluble ( Insoluble ) and –soluble  ( Soluble ) fractions. Cell supernatants ( Medium ) were separated into detergent and aqueous fractions by Triton X-114 phase separation. Samples were analyzed by Western blotting for the presence of FasL using an antibody specific for the extracellular segment of the ligand. The indicated FasL and sFasL  bands were absent in mock transfected controls ( D , and data not shown). The processed sFasL was purified by Fas/Fc affinity chromatography and visualized by Coomassie blue staining ( CB ). ( B ) 293 cells were transfected with mouse FasL cDNA. Total, unfractionated cellular extracts and cell supernatants  were analyzed by Western blotting as described for  A . ( C ) Site of proteinase-induced cleavage of FasL. Metalloproteinase(s) present in 293 cells cleave(s)  membrane-bound FasL at the Ser126–Leu127 bond, liberating a soluble form of FasL. The cleavage site of the TNF-α precursor is shown for comparison. ( D ) The effect of cleavage site mutations on FasL processing. Wild-type ( WT ) FasL as well as FasL mutated at the P1 site ( S126E ), at the P1′ site  ( L127E ), and at both the P1 and P1′ sites ( S126E/L127E ) were transiently transfected into 293T cells. Insoluble proteins (containing membrane-associated FasL) and medium (containing processed sFasL) were analyzed by Western blotting as described above. To achieve similar loading of soluble and  cell-associated FasL, the supernatants corresponding to 10 times more cells were loaded on the gel. A nonspecific band at 60 kD indicates equal loading of  the gel. In the control lane, cell extracts of nontransfected 293 cells were analyzed. ( E ) Activity of naturally processed ( open squares ) and recombinant  ( closed squares ) sFasL on Jurkat T cells.

    Journal: The Journal of Experimental Medicine

    Article Title: Conversion of Membrane-bound Fas(CD95) Ligand to Its Soluble Form Is Associated with Downregulation of Its Proapoptotic Activity and Loss of Liver Toxicity

    doi:

    Figure Lengend Snippet: Recombinant and naturally processed sFasLs show marginal apoptotic activity on human cells. ( A ) Processing of membrane-bound FasL to sFasL. Cellular extracts of a 293 cell clone stably transfected with human FasL cDNA, were separated into Triton X-114–insoluble ( Insoluble ) and –soluble ( Soluble ) fractions. Cell supernatants ( Medium ) were separated into detergent and aqueous fractions by Triton X-114 phase separation. Samples were analyzed by Western blotting for the presence of FasL using an antibody specific for the extracellular segment of the ligand. The indicated FasL and sFasL bands were absent in mock transfected controls ( D , and data not shown). The processed sFasL was purified by Fas/Fc affinity chromatography and visualized by Coomassie blue staining ( CB ). ( B ) 293 cells were transfected with mouse FasL cDNA. Total, unfractionated cellular extracts and cell supernatants were analyzed by Western blotting as described for A . ( C ) Site of proteinase-induced cleavage of FasL. Metalloproteinase(s) present in 293 cells cleave(s) membrane-bound FasL at the Ser126–Leu127 bond, liberating a soluble form of FasL. The cleavage site of the TNF-α precursor is shown for comparison. ( D ) The effect of cleavage site mutations on FasL processing. Wild-type ( WT ) FasL as well as FasL mutated at the P1 site ( S126E ), at the P1′ site ( L127E ), and at both the P1 and P1′ sites ( S126E/L127E ) were transiently transfected into 293T cells. Insoluble proteins (containing membrane-associated FasL) and medium (containing processed sFasL) were analyzed by Western blotting as described above. To achieve similar loading of soluble and cell-associated FasL, the supernatants corresponding to 10 times more cells were loaded on the gel. A nonspecific band at 60 kD indicates equal loading of the gel. In the control lane, cell extracts of nontransfected 293 cells were analyzed. ( E ) Activity of naturally processed ( open squares ) and recombinant ( closed squares ) sFasL on Jurkat T cells.

    Article Snippet: 293T cells transiently transfected with full-length hFasL (wild type and mutants), muFasL, or empty plasmid were transferred in Opti-MEM medium for 5 d. Supernatants were harvested, and the cells were lysed in 2% precondensed Triton X-114 in PBS ( ) containing a protease inhibitor cocktail (Cømplete™; Boehringer Mannheim GmbH , Mannheim, Germany) and subjected to phase separation.

    Techniques: Recombinant, Activity Assay, Stable Transfection, Transfection, Western Blot, Purification, Affinity Chromatography, Staining

    Molecular characterization of the calsenilin knock-out mouse. A , Gene targeting strategy. Top, Wild-type calsenilin locus showing exon 2 and the position of the 5′ and 3′ probes used for genotyping. Bottom, Mutant locus in which exon 2 has been replaced with an IRES, the marker gene β -gal , and the neomycin (neo) resistance gene, the latter under the control of the MC1 promoter. B , Southern blot analysis of genomic DNA from calsenilin +/+, +/-, and -/- mice. Left, Hind III digest; right, Nco I digest. C , Northern blot analysis of mRNA extracted from the brains of calsenilin +/+, +/-, and -/- mice. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is included as a loading control. D ). The asterisk indicates a band migrating just above mouse calsenilin, and the position of IgG (from the immunoprecipitation) is also indicated.

    Journal: The Journal of Neuroscience

    Article Title: Altered Aβ Formation and Long-Term Potentiation in a Calsenilin Knock-Out

    doi: 10.1523/JNEUROSCI.23-27-09097.2003

    Figure Lengend Snippet: Molecular characterization of the calsenilin knock-out mouse. A , Gene targeting strategy. Top, Wild-type calsenilin locus showing exon 2 and the position of the 5′ and 3′ probes used for genotyping. Bottom, Mutant locus in which exon 2 has been replaced with an IRES, the marker gene β -gal , and the neomycin (neo) resistance gene, the latter under the control of the MC1 promoter. B , Southern blot analysis of genomic DNA from calsenilin +/+, +/-, and -/- mice. Left, Hind III digest; right, Nco I digest. C , Northern blot analysis of mRNA extracted from the brains of calsenilin +/+, +/-, and -/- mice. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is included as a loading control. D ). The asterisk indicates a band migrating just above mouse calsenilin, and the position of IgG (from the immunoprecipitation) is also indicated.

    Article Snippet: Cortex, cerebellum, and hippocampus from wild-type and calsenilin knock-out mice were homogenized in immunoprecipitation assay buffer (50 m m Tris, pH 7.4, 150 m m NaCl, 2 m m EDTA, 1% Triton X-100, 1% NP-40, and 0.25% sodium deoxycholate) with protease inhibitors (Complete 1; Boehringer Mannheim, Indianapolis, IN).

    Techniques: Knock-Out, Mutagenesis, Marker, Southern Blot, Mouse Assay, Northern Blot, Immunoprecipitation

    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.

    Journal: Genes & Development

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

    doi:

    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

    (A) Immunoblot analysis of NS1 and its derivatives from primary or persistent JEV infections. Cell lysates from primary infection of BHK-21 cells (lanes 1 and 4) and from persistent infection of B2-5/RP9 (lanes 2 and 5) and C2-2 cells (lanes 3 and 6) were separated by SDS–10% PAGE and immunoblotted with anti-NS1 monoclonal antibody JE7/45-2. Protein samples were treated either with boiling (lanes 4 to 6) or without boiling (lanes 1 to 3). The wild-type NS1 proteins are indicated by arrows. The truncated NS1 and NS1′ are marked by asterisks. Molecular mass markers (kilodaltons) are on the left of the figure. (B) Glycosylation analysis of NS1 proteins by endo-F digestion. Cell lysates from JEV primary infection of BHK-21 (lanes 1 and 4) and from persistent infection of B2-5/RP9 (lanes 2 and 5) and C2-2 cells (lanes 3 and 6) were immunoprecipitated by anti-NS1 monoclonal antibodies and then were treated either without (lanes 1 to 3) or with (lanes 4 to 6) endo-F at 37°C for 16 h. The deglycosylated NS1 and NS1′, as well as their truncated proteins p45 and p39 (lanes 4 to 6), are indicated by open triangles; their counterparts without endo-F digestion (lanes 1 to 3) are marked by arrows. (C) Localization of the truncation region to the N termini of NS1 and NS1′ proteins derived from the persistently JEV-infected cells by immunoprecipitation with anti-NS1 monoclonal antibodies. 35 S-labeled lysates from primarily infected BHK-21 (lanes 1 and 4) and persistently infected B2-1/RP9 (lanes 2 and 5) and B2-5/RP9 (lanes 3 and 6) cells were precipitated by anti-NS1 D2/39.1 (lanes 1 to 3) or JE7/45-2 (lanes 4 to 6) and then separated by SDS–10% PAGE. The wild-type NS1 and NS1′ are marked with arrows, and the truncated protein p39 is marked by an asterisk. Note that only antibody JE7/45-2 recognized the truncated NS1 (lanes 5 and 6).

    Journal: Journal of Virology

    Article Title: Antiapoptotic but Not Antiviral Function of Human bcl-2 Assists Establishment of Japanese Encephalitis Virus Persistence in Cultured Cells

    doi:

    Figure Lengend Snippet: (A) Immunoblot analysis of NS1 and its derivatives from primary or persistent JEV infections. Cell lysates from primary infection of BHK-21 cells (lanes 1 and 4) and from persistent infection of B2-5/RP9 (lanes 2 and 5) and C2-2 cells (lanes 3 and 6) were separated by SDS–10% PAGE and immunoblotted with anti-NS1 monoclonal antibody JE7/45-2. Protein samples were treated either with boiling (lanes 4 to 6) or without boiling (lanes 1 to 3). The wild-type NS1 proteins are indicated by arrows. The truncated NS1 and NS1′ are marked by asterisks. Molecular mass markers (kilodaltons) are on the left of the figure. (B) Glycosylation analysis of NS1 proteins by endo-F digestion. Cell lysates from JEV primary infection of BHK-21 (lanes 1 and 4) and from persistent infection of B2-5/RP9 (lanes 2 and 5) and C2-2 cells (lanes 3 and 6) were immunoprecipitated by anti-NS1 monoclonal antibodies and then were treated either without (lanes 1 to 3) or with (lanes 4 to 6) endo-F at 37°C for 16 h. The deglycosylated NS1 and NS1′, as well as their truncated proteins p45 and p39 (lanes 4 to 6), are indicated by open triangles; their counterparts without endo-F digestion (lanes 1 to 3) are marked by arrows. (C) Localization of the truncation region to the N termini of NS1 and NS1′ proteins derived from the persistently JEV-infected cells by immunoprecipitation with anti-NS1 monoclonal antibodies. 35 S-labeled lysates from primarily infected BHK-21 (lanes 1 and 4) and persistently infected B2-1/RP9 (lanes 2 and 5) and B2-5/RP9 (lanes 3 and 6) cells were precipitated by anti-NS1 D2/39.1 (lanes 1 to 3) or JE7/45-2 (lanes 4 to 6) and then separated by SDS–10% PAGE. The wild-type NS1 and NS1′ are marked with arrows, and the truncated protein p39 is marked by an asterisk. Note that only antibody JE7/45-2 recognized the truncated NS1 (lanes 5 and 6).

    Article Snippet: For endoglycosidase F (endo-F) digestion, the immunoprecipitated proteins were boiled in endo-F boiling buffer (50 mM sodium phosphate, pH 7.5; 0.5% SDS; 1% 2-mercaptoethanol) and then incubated overnight at 37°C with an equal volume of the endo-F incubation buffer (50 mM sodium phosphate, pH 7.5; 2% Nonidet P-40; 0.2% SDS; 1% 2-mercaptoethanol; 25 mM EDTA) with or without endo-F (Boehringer Mannheim).

    Techniques: Infection, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Derivative Assay, Labeling