lysis buffer  (Roche)


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    Structured Review

    Roche lysis buffer
    Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 10808 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lysis buffer/product/Roche
    Average 99 stars, based on 10808 article reviews
    Price from $9.99 to $1999.99
    lysis buffer - by Bioz Stars, 2020-09
    99/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Different isoforms of the Wilms’ tumour protein WT1 have distinct patterns of distribution and trafficking within the nucleus
    Article Snippet: .. For analysis of protein expression, the cells were washed in ice‐cold phosphate buffer saline (PBS) and treated with lysis buffer (10m m Tris‐HCl pH 8.0, 1 m m ethylenediaminetetraacetic acid pH 8.0, 150 m m NaCl and 0.65% NP40) containing a protease inhibitor cocktail (Roche) on ice for 20 min before harvesting and centrifugation at 5000 g for 15 min to remove cell debris. .. Total soluble protein in the cell lysates was measured using the Bio‐Rad protein assay reagent.

    Article Title: SALL4 controls cell fate in response to DNA base composition
    Article Snippet: .. Following a centrifugation for 5min at 1,300rpm, the supernatant was removed and the cell pellet was resuspended in 1ml of lysis buffer (10mM NaCl, 1mM MgCl2, 20mM HEPES pH7.5, 0.1% (v/v) Triton X-100) freshly supplemented with 1x protease inhibitor cocktail (Roche ref. 11873580001) and 0.5mM DTT. .. Supernatant was removed and nuclei were resuspended in 1ml of lysis buffer freshly supplemented with 1x protease inhibitor cocktail and 0.5mM DTT.

    Article Title: JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton
    Article Snippet: .. After post-differentiation in cortical media, i3Neurons (typically 17 days post-differentiation) were washed with ice-cold PBS and then lysed in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) supplemented with cOmplete™ EDTA-free protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche), followed by centrifugation at 13,000xg for 6 min. .. The supernatant was collected and incubated at 95°C for 5 min in SDS sample buffer containing 1% 2-mercaptoethanol (Sigma).

    Protease Inhibitor:

    Article Title: Genome modularization reveals overlapped gene topology is necessary for efficient viral reproduction
    Article Snippet: .. The next day, sample pellets were resuspended in 200 µL lysis buffer (100 mM triethylammonium bicarbonate buffer (TEAB) with 1% (v/v) sodium deoxycholate (SDC) and 1x protease inhibitor cocktail solution (Roche, Switzerland)), and subjected to probe sonication (30% amplitude, 5 bursts). .. Reduction and alkylation of cysteine residues (dithiothreitol (DTT) to 10 mM, incubated at 60°C for 30-minutes followed by iodoacetamide (IAA) to 30 mM, incubated 1-hour in the dark, and lastly, quenching with equal molarity of DTT), followed by chloroform-methanol protein precipitation to obtain pure protein pellets that were subsequently resuspended in digestion buffer (100 mM TEAB with 1% (v/v) SDC) and quantified by the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, United States).

    Article Title: Different isoforms of the Wilms’ tumour protein WT1 have distinct patterns of distribution and trafficking within the nucleus
    Article Snippet: .. For analysis of protein expression, the cells were washed in ice‐cold phosphate buffer saline (PBS) and treated with lysis buffer (10m m Tris‐HCl pH 8.0, 1 m m ethylenediaminetetraacetic acid pH 8.0, 150 m m NaCl and 0.65% NP40) containing a protease inhibitor cocktail (Roche) on ice for 20 min before harvesting and centrifugation at 5000 g for 15 min to remove cell debris. .. Total soluble protein in the cell lysates was measured using the Bio‐Rad protein assay reagent.

    Article Title: Synaptic anchoring of the endoplasmic reticulum depends on myosin V and caldendrin activity
    Article Snippet: .. For preparation of whole mouse brain extract, 9 ml lysis buffer (50mM Tris HCl pH 7.4, 150 mM NaCl, 0.1 % SDS, 0.2 % NP-40, complete protease inhibitor cocktail (Roche )) were added per 1 g of tissue weight, and the tissue was lysed using a Dounce homogenizer. .. The washed beads were then combined with 1 ml of the cleared mouse brain lysate and incubated for 1 h at 4 °C on a rotator.

    Article Title: SALL4 controls cell fate in response to DNA base composition
    Article Snippet: .. Following a centrifugation for 5min at 1,300rpm, the supernatant was removed and the cell pellet was resuspended in 1ml of lysis buffer (10mM NaCl, 1mM MgCl2, 20mM HEPES pH7.5, 0.1% (v/v) Triton X-100) freshly supplemented with 1x protease inhibitor cocktail (Roche ref. 11873580001) and 0.5mM DTT. .. Supernatant was removed and nuclei were resuspended in 1ml of lysis buffer freshly supplemented with 1x protease inhibitor cocktail and 0.5mM DTT.

    Article Title: Temporal modulation of the NF-κB RelA network in response to different types of DNA damage
    Article Snippet: .. After removal of the media, cells were washed three times with ice cold PBS and collected in 400 μL of lysis buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) Triton X-100, 0.5% (v/v) NP-40, 1 x PhosSTOP™ phosphatase inhibitor cocktail tablet (Roche), 1 x cOmplete™ Protease Inhibitor Cocktail (Roche), 50 U/mL of benzonase) into 1.5 mL microtubes using a cell scraper. .. Cells were then incubated on ice for 120 min to permit cell lysis to occur.

    Article Title: JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton
    Article Snippet: .. After post-differentiation in cortical media, i3Neurons (typically 17 days post-differentiation) were washed with ice-cold PBS and then lysed in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) supplemented with cOmplete™ EDTA-free protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche), followed by centrifugation at 13,000xg for 6 min. .. The supernatant was collected and incubated at 95°C for 5 min in SDS sample buffer containing 1% 2-mercaptoethanol (Sigma).

    Article Title: Reprogramming of stromal fibroblasts by chemotherapy-induced secretion of IFNβ1 drives re-growth of breast cancer cells after treatment
    Article Snippet: .. Cell pellets were lysed in Lysis Buffer containing 20 mM Tris/HCl (pH7.5), 150 mM NaCl, 1% Triton X-100, 1 mM DTT, 2 mM EDTA, 1 mM NaF, 1 mM Ortho-Vanadate, 25 mM beta-glycerophosphate and protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). .. Protein concentration of the samples was measured with BCA protein assay kit (Thermo Fisher Scientific, Massachusetts, USA) and quantified with GloMax microplate reader (Promega GmbH, Walldorf, Germany).

    Article Title: UvrC Coordinates an O2-Sensitive [4Fe4S] Cofactor
    Article Snippet: .. While columns were equilibrating, cell pellets were thawed, resuspended in Lysis Buffer (100 mL lysis buffer per 10 g of wet pellet) that was supplemented on the day of the purification with 6–8 tablets of crushed cOmplete protease inhibitor cocktail tablets (Roche), DNase (15 kU, Sigma), and DTT (1 mM), and homogenized on ice using a Dounce homogenizer. .. The cell slurry was passed over a 100 μm nylon cell strainer (Corning) and l ysed using an Emulsiflex-C5 (Avestin) homogenizer at 25 000 psi under a positive pressure of Ar over two cycles.

    Purification:

    Article Title: UvrC Coordinates an O2-Sensitive [4Fe4S] Cofactor
    Article Snippet: .. While columns were equilibrating, cell pellets were thawed, resuspended in Lysis Buffer (100 mL lysis buffer per 10 g of wet pellet) that was supplemented on the day of the purification with 6–8 tablets of crushed cOmplete protease inhibitor cocktail tablets (Roche), DNase (15 kU, Sigma), and DTT (1 mM), and homogenized on ice using a Dounce homogenizer. .. The cell slurry was passed over a 100 μm nylon cell strainer (Corning) and l ysed using an Emulsiflex-C5 (Avestin) homogenizer at 25 000 psi under a positive pressure of Ar over two cycles.

    Expressing:

    Article Title: Different isoforms of the Wilms’ tumour protein WT1 have distinct patterns of distribution and trafficking within the nucleus
    Article Snippet: .. For analysis of protein expression, the cells were washed in ice‐cold phosphate buffer saline (PBS) and treated with lysis buffer (10m m Tris‐HCl pH 8.0, 1 m m ethylenediaminetetraacetic acid pH 8.0, 150 m m NaCl and 0.65% NP40) containing a protease inhibitor cocktail (Roche) on ice for 20 min before harvesting and centrifugation at 5000 g for 15 min to remove cell debris. .. Total soluble protein in the cell lysates was measured using the Bio‐Rad protein assay reagent.

    Sonication:

    Article Title: Genome modularization reveals overlapped gene topology is necessary for efficient viral reproduction
    Article Snippet: .. The next day, sample pellets were resuspended in 200 µL lysis buffer (100 mM triethylammonium bicarbonate buffer (TEAB) with 1% (v/v) sodium deoxycholate (SDC) and 1x protease inhibitor cocktail solution (Roche, Switzerland)), and subjected to probe sonication (30% amplitude, 5 bursts). .. Reduction and alkylation of cysteine residues (dithiothreitol (DTT) to 10 mM, incubated at 60°C for 30-minutes followed by iodoacetamide (IAA) to 30 mM, incubated 1-hour in the dark, and lastly, quenching with equal molarity of DTT), followed by chloroform-methanol protein precipitation to obtain pure protein pellets that were subsequently resuspended in digestion buffer (100 mM TEAB with 1% (v/v) SDC) and quantified by the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, United States).

    Lysis:

    Article Title: Genome modularization reveals overlapped gene topology is necessary for efficient viral reproduction
    Article Snippet: .. The next day, sample pellets were resuspended in 200 µL lysis buffer (100 mM triethylammonium bicarbonate buffer (TEAB) with 1% (v/v) sodium deoxycholate (SDC) and 1x protease inhibitor cocktail solution (Roche, Switzerland)), and subjected to probe sonication (30% amplitude, 5 bursts). .. Reduction and alkylation of cysteine residues (dithiothreitol (DTT) to 10 mM, incubated at 60°C for 30-minutes followed by iodoacetamide (IAA) to 30 mM, incubated 1-hour in the dark, and lastly, quenching with equal molarity of DTT), followed by chloroform-methanol protein precipitation to obtain pure protein pellets that were subsequently resuspended in digestion buffer (100 mM TEAB with 1% (v/v) SDC) and quantified by the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, United States).

    Article Title: Different isoforms of the Wilms’ tumour protein WT1 have distinct patterns of distribution and trafficking within the nucleus
    Article Snippet: .. For analysis of protein expression, the cells were washed in ice‐cold phosphate buffer saline (PBS) and treated with lysis buffer (10m m Tris‐HCl pH 8.0, 1 m m ethylenediaminetetraacetic acid pH 8.0, 150 m m NaCl and 0.65% NP40) containing a protease inhibitor cocktail (Roche) on ice for 20 min before harvesting and centrifugation at 5000 g for 15 min to remove cell debris. .. Total soluble protein in the cell lysates was measured using the Bio‐Rad protein assay reagent.

    Article Title: Synaptic anchoring of the endoplasmic reticulum depends on myosin V and caldendrin activity
    Article Snippet: .. For preparation of whole mouse brain extract, 9 ml lysis buffer (50mM Tris HCl pH 7.4, 150 mM NaCl, 0.1 % SDS, 0.2 % NP-40, complete protease inhibitor cocktail (Roche )) were added per 1 g of tissue weight, and the tissue was lysed using a Dounce homogenizer. .. The washed beads were then combined with 1 ml of the cleared mouse brain lysate and incubated for 1 h at 4 °C on a rotator.

    Article Title: SALL4 controls cell fate in response to DNA base composition
    Article Snippet: .. Following a centrifugation for 5min at 1,300rpm, the supernatant was removed and the cell pellet was resuspended in 1ml of lysis buffer (10mM NaCl, 1mM MgCl2, 20mM HEPES pH7.5, 0.1% (v/v) Triton X-100) freshly supplemented with 1x protease inhibitor cocktail (Roche ref. 11873580001) and 0.5mM DTT. .. Supernatant was removed and nuclei were resuspended in 1ml of lysis buffer freshly supplemented with 1x protease inhibitor cocktail and 0.5mM DTT.

    Article Title: Temporal modulation of the NF-κB RelA network in response to different types of DNA damage
    Article Snippet: .. After removal of the media, cells were washed three times with ice cold PBS and collected in 400 μL of lysis buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) Triton X-100, 0.5% (v/v) NP-40, 1 x PhosSTOP™ phosphatase inhibitor cocktail tablet (Roche), 1 x cOmplete™ Protease Inhibitor Cocktail (Roche), 50 U/mL of benzonase) into 1.5 mL microtubes using a cell scraper. .. Cells were then incubated on ice for 120 min to permit cell lysis to occur.

    Article Title: JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton
    Article Snippet: .. After post-differentiation in cortical media, i3Neurons (typically 17 days post-differentiation) were washed with ice-cold PBS and then lysed in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) supplemented with cOmplete™ EDTA-free protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche), followed by centrifugation at 13,000xg for 6 min. .. The supernatant was collected and incubated at 95°C for 5 min in SDS sample buffer containing 1% 2-mercaptoethanol (Sigma).

    Article Title: Reprogramming of stromal fibroblasts by chemotherapy-induced secretion of IFNβ1 drives re-growth of breast cancer cells after treatment
    Article Snippet: .. Cell pellets were lysed in Lysis Buffer containing 20 mM Tris/HCl (pH7.5), 150 mM NaCl, 1% Triton X-100, 1 mM DTT, 2 mM EDTA, 1 mM NaF, 1 mM Ortho-Vanadate, 25 mM beta-glycerophosphate and protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). .. Protein concentration of the samples was measured with BCA protein assay kit (Thermo Fisher Scientific, Massachusetts, USA) and quantified with GloMax microplate reader (Promega GmbH, Walldorf, Germany).

    Article Title: UvrC Coordinates an O2-Sensitive [4Fe4S] Cofactor
    Article Snippet: .. While columns were equilibrating, cell pellets were thawed, resuspended in Lysis Buffer (100 mL lysis buffer per 10 g of wet pellet) that was supplemented on the day of the purification with 6–8 tablets of crushed cOmplete protease inhibitor cocktail tablets (Roche), DNase (15 kU, Sigma), and DTT (1 mM), and homogenized on ice using a Dounce homogenizer. .. The cell slurry was passed over a 100 μm nylon cell strainer (Corning) and l ysed using an Emulsiflex-C5 (Avestin) homogenizer at 25 000 psi under a positive pressure of Ar over two cycles.

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  • 89
    Roche flag lysis buffer
    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with <t>anti-FLAG</t> antibodies, resolved by SDS-PAGE, and detected by western blotting using the <t>M2</t> anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.
    Flag Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flag lysis buffer/product/Roche
    Average 89 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
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    80
    merck kgaa tube lysis buffer
    Characterisation of the RNF26 ubiquitin ligase complex. ( a ) Domain organisation of human RNF26 protein. ( b ) Protein sequence alignment of the RING domain and C-terminus of RNF26 with those of human RNF4 (P78317), XIAP (P98170), BIRC2 (Q13490) and MDM2 (Q00987). Conserved residues are demarcated according to the Rasmol colour scheme. ( c ) DOX titration of Flp-In ™ 293 cells stably expressing FH-RNF26 WT or FH-RNF26 Y432A ± <t>MG132</t> with lysates separated by SDS-PAGE and resulting western blots probed for RNF26 (anti-HA) and tubulin. ( d ) 35 S-Met/Cys pulse-chase assay (0, 1, 2 h) of DOX-induced Flp-In ™ 293 cells stably expressing FH-RNF26 WT or FH-RNF26 Y432A , ± MG132 and immunoprecipitated by anti-FLAG agarose. ( e ) Co-immunoprecipitation of endogenous RNF26 from DOX-induced Flp-In ™ 293 cells stably expressing FH-RNF26 WT or FH-RNF26 Y432A by anti-FLAG beads. Detection of FLAG-HA (FH) and endogenous (e) RNF26 by western blot using t he indicated antibodies. ( f ) <t>TUBE</t> pulldowns from FH-RNF26 WT or FH-RNF26 Y432A Flp-In ™ 293 cell lysates, ± MG132 and Usp21.
    Tube Lysis Buffer, supplied by merck kgaa, used in various techniques. Bioz Stars score: 80/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 80 stars, based on 1 article reviews
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    90
    Roche immunoprecipitation ip lysis buffer
    The ubiquitination of NS4A at Lys132 is important for the interaction of NS4A and STAT1. (A) HEK293 T cells (1 × 10 6 ) were transfected with the indicated plasmids. 24 h after transfection, ubiquitination assays and immunoblotting analysis were performed with the indicated antibodies. (B) Effect of K132R mutant on ubiquitination of NS4A. The experiments were performed as in A except that the indicated plasmids were used. (C) Effect of K132R mutant on the interaction of NS4A with STAT1. HEK293 T cells (1 × 10 6 ) transfected with the indicated plasmids (1 μg each) for 36 h followed by <t>co-immunoprecipitation</t> experiments and immunoblotting analysis with the indicated antibodies. (D) Effect of K132R mutant on the dimer of STAT1 and STAT2. The cells were performed as in C. The dimer of STAT1/2 was analysed by Native PAGE. (E) Effect of NS4A peptide on the interaction of STAT1 and STAT2. HEK293 T cells (1 × 10 6 ) were transfected with Flag-STAT1 and HA-STAT2. 48 h after transfection, the cells were lysed and then added the different peptides of NS4A, IP analyses the interaction of STAT1 with STAT2. (F G) Immunoblot analysis of the ubiquitination of NS4A in T98G cells co-transfected with HA-Ub, HA-Ub-K27R (F) or HA-Ub-K27O (G). Data are representative of three independent experiments.
    Immunoprecipitation Ip Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche deoxycholate doc lysis buffer
    Increased acetylation promotes fibronectin (FN) matrix assembly. ( A ) Mesangial cells grown in 5 mM or 30 mM glucose for 48 h were lysed in RIPA buffer. ( B , C ) Mesangial cells grown in 30 mM glucose were treated with either 5 μM suberoylanilide hydroxamic acid (SAHA) or suberohydroxamic acid (SBHA) or DMSO (vehicle control) in medium containing 20 μg/mL human plasma FN for 24 h before lysis in <t>deoxycholate</t> <t>(DOC)</t> buffer. ( A , B ) RIPA and DOC-soluble lysates were immunoblotted with antibodies against acetyl-tubulin (Ac-Tub), tubulin (Tub) or GAPDH as indicated. ( C ) The DOC-insoluble fraction was immunoblotted with HFN7.1 anti-FN monoclonal antibody. Fold-changes are the average of three independent experiments ( p
    Deoxycholate Doc Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deoxycholate doc lysis buffer/product/Roche
    Average 89 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
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    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.

    Journal: Journal of viral hepatitis

    Article Title: Sequences in the terminal protein and reverse transcriptase domains of the Hepatitis B Virus polymerase contribute to RNA binding and encapsidation

    doi: 10.1111/jvh.12225

    Figure Lengend Snippet: In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.

    Article Snippet: The FLAG lysis buffer was removed from aliquots of P-bound M2 beads, and then aliquots of beads were incubated with 0.5 μg 32 P-labeled ε RNAs in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA, 0.05% NP-40] with 1× complete protease inhibitor cocktail (Roche), 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 U/μl RNasin Plus RNase inhibitor (Promega) ( ).

    Techniques: In Vitro, RNA Binding Assay, Activity Assay, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Western Blot, Purification, Incubation, Labeling, Binding Assay, Standard Deviation

    Characterisation of the RNF26 ubiquitin ligase complex. ( a ) Domain organisation of human RNF26 protein. ( b ) Protein sequence alignment of the RING domain and C-terminus of RNF26 with those of human RNF4 (P78317), XIAP (P98170), BIRC2 (Q13490) and MDM2 (Q00987). Conserved residues are demarcated according to the Rasmol colour scheme. ( c ) DOX titration of Flp-In ™ 293 cells stably expressing FH-RNF26 WT or FH-RNF26 Y432A ± MG132 with lysates separated by SDS-PAGE and resulting western blots probed for RNF26 (anti-HA) and tubulin. ( d ) 35 S-Met/Cys pulse-chase assay (0, 1, 2 h) of DOX-induced Flp-In ™ 293 cells stably expressing FH-RNF26 WT or FH-RNF26 Y432A , ± MG132 and immunoprecipitated by anti-FLAG agarose. ( e ) Co-immunoprecipitation of endogenous RNF26 from DOX-induced Flp-In ™ 293 cells stably expressing FH-RNF26 WT or FH-RNF26 Y432A by anti-FLAG beads. Detection of FLAG-HA (FH) and endogenous (e) RNF26 by western blot using t he indicated antibodies. ( f ) TUBE pulldowns from FH-RNF26 WT or FH-RNF26 Y432A Flp-In ™ 293 cell lysates, ± MG132 and Usp21.

    Journal: bioRxiv

    Article Title: Interaction mapping of endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signalling

    doi: 10.1101/2020.03.18.993998

    Figure Lengend Snippet: Characterisation of the RNF26 ubiquitin ligase complex. ( a ) Domain organisation of human RNF26 protein. ( b ) Protein sequence alignment of the RING domain and C-terminus of RNF26 with those of human RNF4 (P78317), XIAP (P98170), BIRC2 (Q13490) and MDM2 (Q00987). Conserved residues are demarcated according to the Rasmol colour scheme. ( c ) DOX titration of Flp-In ™ 293 cells stably expressing FH-RNF26 WT or FH-RNF26 Y432A ± MG132 with lysates separated by SDS-PAGE and resulting western blots probed for RNF26 (anti-HA) and tubulin. ( d ) 35 S-Met/Cys pulse-chase assay (0, 1, 2 h) of DOX-induced Flp-In ™ 293 cells stably expressing FH-RNF26 WT or FH-RNF26 Y432A , ± MG132 and immunoprecipitated by anti-FLAG agarose. ( e ) Co-immunoprecipitation of endogenous RNF26 from DOX-induced Flp-In ™ 293 cells stably expressing FH-RNF26 WT or FH-RNF26 Y432A by anti-FLAG beads. Detection of FLAG-HA (FH) and endogenous (e) RNF26 by western blot using t he indicated antibodies. ( f ) TUBE pulldowns from FH-RNF26 WT or FH-RNF26 Y432A Flp-In ™ 293 cell lysates, ± MG132 and Usp21.

    Article Snippet: Immuno- and affinity purification of ubiquitinated proteins FH-RNF26WT and FH-RNF26Y432A -expressing Flp-In™ 293 cells were induced with DOX (18 h) and where indicated, samples were additionally treated with 10 μM MG132 for 2 h. Cell pellets were lysed in TUBE lysis buffer (20 mM sodium phosphate pH 7.5, 1% NP-40 (v/v), 2 mM EDTA, supplemented with cOmplete protease inhibitor cocktail (Roche), PhosSTOP (Roche), NEM (50 mM), and DTT (1 mM)).

    Techniques: Sequencing, Titration, Stable Transfection, Expressing, SDS Page, Western Blot, Pulse Chase, Immunoprecipitation

    The ubiquitination of NS4A at Lys132 is important for the interaction of NS4A and STAT1. (A) HEK293 T cells (1 × 10 6 ) were transfected with the indicated plasmids. 24 h after transfection, ubiquitination assays and immunoblotting analysis were performed with the indicated antibodies. (B) Effect of K132R mutant on ubiquitination of NS4A. The experiments were performed as in A except that the indicated plasmids were used. (C) Effect of K132R mutant on the interaction of NS4A with STAT1. HEK293 T cells (1 × 10 6 ) transfected with the indicated plasmids (1 μg each) for 36 h followed by co-immunoprecipitation experiments and immunoblotting analysis with the indicated antibodies. (D) Effect of K132R mutant on the dimer of STAT1 and STAT2. The cells were performed as in C. The dimer of STAT1/2 was analysed by Native PAGE. (E) Effect of NS4A peptide on the interaction of STAT1 and STAT2. HEK293 T cells (1 × 10 6 ) were transfected with Flag-STAT1 and HA-STAT2. 48 h after transfection, the cells were lysed and then added the different peptides of NS4A, IP analyses the interaction of STAT1 with STAT2. (F G) Immunoblot analysis of the ubiquitination of NS4A in T98G cells co-transfected with HA-Ub, HA-Ub-K27R (F) or HA-Ub-K27O (G). Data are representative of three independent experiments.

    Journal: Emerging Microbes & Infections

    Article Title: Tick-borne encephalitis virus NS4A ubiquitination antagonizes type I interferon-stimulated STAT1/2 signalling pathway

    doi: 10.1080/22221751.2020.1745094

    Figure Lengend Snippet: The ubiquitination of NS4A at Lys132 is important for the interaction of NS4A and STAT1. (A) HEK293 T cells (1 × 10 6 ) were transfected with the indicated plasmids. 24 h after transfection, ubiquitination assays and immunoblotting analysis were performed with the indicated antibodies. (B) Effect of K132R mutant on ubiquitination of NS4A. The experiments were performed as in A except that the indicated plasmids were used. (C) Effect of K132R mutant on the interaction of NS4A with STAT1. HEK293 T cells (1 × 10 6 ) transfected with the indicated plasmids (1 μg each) for 36 h followed by co-immunoprecipitation experiments and immunoblotting analysis with the indicated antibodies. (D) Effect of K132R mutant on the dimer of STAT1 and STAT2. The cells were performed as in C. The dimer of STAT1/2 was analysed by Native PAGE. (E) Effect of NS4A peptide on the interaction of STAT1 and STAT2. HEK293 T cells (1 × 10 6 ) were transfected with Flag-STAT1 and HA-STAT2. 48 h after transfection, the cells were lysed and then added the different peptides of NS4A, IP analyses the interaction of STAT1 with STAT2. (F G) Immunoblot analysis of the ubiquitination of NS4A in T98G cells co-transfected with HA-Ub, HA-Ub-K27R (F) or HA-Ub-K27O (G). Data are representative of three independent experiments.

    Article Snippet: Coimmunoprecipitations, Western blotting, and ubiquitination assays Whole-cell lysates were prepared 36 h post-transient transfection in a immunoprecipitation (IP) lysis buffer containing 50 mM Tris, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 150 mM NaCl, 100 µM phenylmethylsulfonyl fluoride (PMSF) and Complete TM protease inhibitors (Roche Applied Science) for 30 min in 4°C.

    Techniques: Transfection, Mutagenesis, Immunoprecipitation, Clear Native PAGE

    Increased acetylation promotes fibronectin (FN) matrix assembly. ( A ) Mesangial cells grown in 5 mM or 30 mM glucose for 48 h were lysed in RIPA buffer. ( B , C ) Mesangial cells grown in 30 mM glucose were treated with either 5 μM suberoylanilide hydroxamic acid (SAHA) or suberohydroxamic acid (SBHA) or DMSO (vehicle control) in medium containing 20 μg/mL human plasma FN for 24 h before lysis in deoxycholate (DOC) buffer. ( A , B ) RIPA and DOC-soluble lysates were immunoblotted with antibodies against acetyl-tubulin (Ac-Tub), tubulin (Tub) or GAPDH as indicated. ( C ) The DOC-insoluble fraction was immunoblotted with HFN7.1 anti-FN monoclonal antibody. Fold-changes are the average of three independent experiments ( p

    Journal: Cells

    Article Title: Stimulation of Fibronectin Matrix Assembly by Lysine Acetylation

    doi: 10.3390/cells9030655

    Figure Lengend Snippet: Increased acetylation promotes fibronectin (FN) matrix assembly. ( A ) Mesangial cells grown in 5 mM or 30 mM glucose for 48 h were lysed in RIPA buffer. ( B , C ) Mesangial cells grown in 30 mM glucose were treated with either 5 μM suberoylanilide hydroxamic acid (SAHA) or suberohydroxamic acid (SBHA) or DMSO (vehicle control) in medium containing 20 μg/mL human plasma FN for 24 h before lysis in deoxycholate (DOC) buffer. ( A , B ) RIPA and DOC-soluble lysates were immunoblotted with antibodies against acetyl-tubulin (Ac-Tub), tubulin (Tub) or GAPDH as indicated. ( C ) The DOC-insoluble fraction was immunoblotted with HFN7.1 anti-FN monoclonal antibody. Fold-changes are the average of three independent experiments ( p

    Article Snippet: Confluent cells were washed with cold phosphate buffered saline (PBS) and then lysed in deoxycholate (DOC) lysis buffer (2% DOC, 20mM Tris-HCl, pH 8.8, 2 mM EDTA, and protease inhibitor cocktail (Roche)) according to our standard procedure [ ].

    Techniques: Lysis