Structured Review

Roche nonidet p40
Substitution of Lys-110 with an alanine residue results in the accumulation of the mutant PCNA in the heterochromatin region ( A ) The cell fractionation protocol. CHO cells transfected with WT or mutant constructs were fractionated at 12 h post-transfection. MNase, micrococcal nuclease; NP-40, <t>Nonidet</t> <t>P40;</t> Sup, supernatant. ( B ) Fractionated samples were analysed by SDS/PAGE and Western blotting with anti-GFP, -PCNA, -(histone H1) or -HP1α antibodies as indicated. The striking differences in chromatin localization of PCNA(K110A) and endogenous PCNA are shown with * and arrowheads. ( C ) DNA purified from each fraction was separated by agarose gel electrophoresis (2% gel). Di, di-nucleosome; M, DNA size makers; Mono, mononucleosome.
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Images

1) Product Images from "Lys-110 is essential for targeting PCNA to replication and repair foci, and the K110A mutant activates apoptosis"

Article Title: Lys-110 is essential for targeting PCNA to replication and repair foci, and the K110A mutant activates apoptosis

Journal: Biology of the cell

doi: 10.1042/BC20070158

Substitution of Lys-110 with an alanine residue results in the accumulation of the mutant PCNA in the heterochromatin region ( A ) The cell fractionation protocol. CHO cells transfected with WT or mutant constructs were fractionated at 12 h post-transfection. MNase, micrococcal nuclease; NP-40, Nonidet P40; Sup, supernatant. ( B ) Fractionated samples were analysed by SDS/PAGE and Western blotting with anti-GFP, -PCNA, -(histone H1) or -HP1α antibodies as indicated. The striking differences in chromatin localization of PCNA(K110A) and endogenous PCNA are shown with * and arrowheads. ( C ) DNA purified from each fraction was separated by agarose gel electrophoresis (2% gel). Di, di-nucleosome; M, DNA size makers; Mono, mononucleosome.
Figure Legend Snippet: Substitution of Lys-110 with an alanine residue results in the accumulation of the mutant PCNA in the heterochromatin region ( A ) The cell fractionation protocol. CHO cells transfected with WT or mutant constructs were fractionated at 12 h post-transfection. MNase, micrococcal nuclease; NP-40, Nonidet P40; Sup, supernatant. ( B ) Fractionated samples were analysed by SDS/PAGE and Western blotting with anti-GFP, -PCNA, -(histone H1) or -HP1α antibodies as indicated. The striking differences in chromatin localization of PCNA(K110A) and endogenous PCNA are shown with * and arrowheads. ( C ) DNA purified from each fraction was separated by agarose gel electrophoresis (2% gel). Di, di-nucleosome; M, DNA size makers; Mono, mononucleosome.

Techniques Used: Mutagenesis, Cell Fractionation, Transfection, Construct, SDS Page, Western Blot, Purification, Agarose Gel Electrophoresis

2) Product Images from "CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells"

Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells

Journal: Biochemical Journal

doi: 10.1042/BJ20040741

Detection of intracellular CD83 in monocytes, macrophages and imDCs ( A ) Monocytes (Mo), macrophages (MΦ) and imDCs were fixed with paraformaldehyde and permeabilized with saponin. Upon blocking with 20% (v/v) goat serum, the cells were stained with a PE-labelled CD83 antibody (clone HB15e; filled histograms) or, as a control, isotype IgG (open histograms). The monocytic THP-1 cells were used as controls. ( B ) Monocytes (lane 3), resting macrophages (lane 4), LPS-activated macrophages (lane 5), imDCs (lane 6) and mDCs (lane 7) were lysed with Nonidet P40, and the soluble fractions were analysed by Western blotting using the HB15a CD83 antibody (Santa Cruz Biotechnology). Aliquots (50 μg) of each sample were separated by SDS/PAGE. Also included in the blots was the soluble fraction of THP-1 cells (lane 8), and 293T cells transfected with the pcDNA3.1 plasmid (lane 1) or the phCD83 expression vector (lane 2). The transfected cells were cultured for 24 h. For monocytes, both the soluble (lane 9) and insoluble (lane 10) fractions were subjected to Western blot analysis. ( C ) Expression of CD83 by 293T cells: 293T cells were transfected with the phCD83 expression vector (lower panel) or, as a control, with the pcDNA3.1 plasmid (upper panel). The cells were stained with a PE-labelled CD83 antibody (HB15e; open histograms). The filled histograms represent signals detected with isotype IgG. All results shown are representative of at least three similar experiments.
Figure Legend Snippet: Detection of intracellular CD83 in monocytes, macrophages and imDCs ( A ) Monocytes (Mo), macrophages (MΦ) and imDCs were fixed with paraformaldehyde and permeabilized with saponin. Upon blocking with 20% (v/v) goat serum, the cells were stained with a PE-labelled CD83 antibody (clone HB15e; filled histograms) or, as a control, isotype IgG (open histograms). The monocytic THP-1 cells were used as controls. ( B ) Monocytes (lane 3), resting macrophages (lane 4), LPS-activated macrophages (lane 5), imDCs (lane 6) and mDCs (lane 7) were lysed with Nonidet P40, and the soluble fractions were analysed by Western blotting using the HB15a CD83 antibody (Santa Cruz Biotechnology). Aliquots (50 μg) of each sample were separated by SDS/PAGE. Also included in the blots was the soluble fraction of THP-1 cells (lane 8), and 293T cells transfected with the pcDNA3.1 plasmid (lane 1) or the phCD83 expression vector (lane 2). The transfected cells were cultured for 24 h. For monocytes, both the soluble (lane 9) and insoluble (lane 10) fractions were subjected to Western blot analysis. ( C ) Expression of CD83 by 293T cells: 293T cells were transfected with the phCD83 expression vector (lower panel) or, as a control, with the pcDNA3.1 plasmid (upper panel). The cells were stained with a PE-labelled CD83 antibody (HB15e; open histograms). The filled histograms represent signals detected with isotype IgG. All results shown are representative of at least three similar experiments.

Techniques Used: Blocking Assay, Staining, Western Blot, SDS Page, Transfection, Plasmid Preparation, Expressing, Cell Culture

Surface induction of CD83 on DCs requires Asn-linked glycosylation ( A ) Transfected 293T cells were lysed, and the soluble fraction was either untreated (lane 1) or treated with PNGase F (lane 2). The samples were then separated on SDS/PAGE gels and subjected to Western blotting using the HB15a CD83 antibody. The transfected 293T cells were also cultured in the presence of tunicamycin (lane 3) or DMSO (lane 4) for 24 h, and then lysed with Nonidet P40. The soluble fraction was subjected to SDS/PAGE, followed by Western blotting using the CD83 antibody. ImDCs were activated with LPS in the presence of tunicamycin (lane 5) or DMSO (lane 6) for 6 h, and were similarly analysed by Western blotting using the CD83 antibody. The molecular-mass standards are shown, and apply to all three blots. ( B ) Transfected 293T cells were cultured for 24 h in the presence of tunicamycin (solid line) or DMSO (dotted line), and then stained with the CD83 antibody and analysed by flow cytometry. The filled histogram represents signals on transfected 293T cells cultured in the absence of tunicamycin and DMSO. ( C ) ImDCs were activated with LPS in the presence of tunicamycin (solid line) or DMSO (dotted line), or were unactivated (filled histogram) and then stained with the CD83 antibody and analysed by flow cytometry. The results are representative of two or three independent experiments.
Figure Legend Snippet: Surface induction of CD83 on DCs requires Asn-linked glycosylation ( A ) Transfected 293T cells were lysed, and the soluble fraction was either untreated (lane 1) or treated with PNGase F (lane 2). The samples were then separated on SDS/PAGE gels and subjected to Western blotting using the HB15a CD83 antibody. The transfected 293T cells were also cultured in the presence of tunicamycin (lane 3) or DMSO (lane 4) for 24 h, and then lysed with Nonidet P40. The soluble fraction was subjected to SDS/PAGE, followed by Western blotting using the CD83 antibody. ImDCs were activated with LPS in the presence of tunicamycin (lane 5) or DMSO (lane 6) for 6 h, and were similarly analysed by Western blotting using the CD83 antibody. The molecular-mass standards are shown, and apply to all three blots. ( B ) Transfected 293T cells were cultured for 24 h in the presence of tunicamycin (solid line) or DMSO (dotted line), and then stained with the CD83 antibody and analysed by flow cytometry. The filled histogram represents signals on transfected 293T cells cultured in the absence of tunicamycin and DMSO. ( C ) ImDCs were activated with LPS in the presence of tunicamycin (solid line) or DMSO (dotted line), or were unactivated (filled histogram) and then stained with the CD83 antibody and analysed by flow cytometry. The results are representative of two or three independent experiments.

Techniques Used: Transfection, SDS Page, Western Blot, Cell Culture, Staining, Flow Cytometry, Cytometry

3) Product Images from "Evaluation of Soluble Junctional Adhesion Molecule-A as a Biomarker of Human Brain Endothelial Barrier Breakdown"

Article Title: Evaluation of Soluble Junctional Adhesion Molecule-A as a Biomarker of Human Brain Endothelial Barrier Breakdown

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013568

Pro-inflammatory stimulation of HBMEC induces JAM-A dissociation from the actin cytoskeleton. A Primary HBMEC were either left unstimulated or treated with TNF-α 10 ng/mL or IFN-γ 100 IU/mL alone or in combination. Cell protein extracts were generated with a Nonidet-P40 based cell lysis buffer and subjected to Western blot analysis. JAM-A was stained with M.Ab.F11. Staining of the same, peroxidase-inactivated membranes with a rabbit antibody against β-actin served as a loading control. B N-deglycosylation of JAM-A with increasing concentrations of PNGase F for 2 h. A rabbit polyclonal antibody against JAM-A (Zymed) was used for the detection of JAM-A. The asterisk represents N-glycosylated, the open circle N-declycosylated JAM-A. Representative experiments out of at least 5 independent experiments with different EC preparations for each subpanel of the figure are shown.
Figure Legend Snippet: Pro-inflammatory stimulation of HBMEC induces JAM-A dissociation from the actin cytoskeleton. A Primary HBMEC were either left unstimulated or treated with TNF-α 10 ng/mL or IFN-γ 100 IU/mL alone or in combination. Cell protein extracts were generated with a Nonidet-P40 based cell lysis buffer and subjected to Western blot analysis. JAM-A was stained with M.Ab.F11. Staining of the same, peroxidase-inactivated membranes with a rabbit antibody against β-actin served as a loading control. B N-deglycosylation of JAM-A with increasing concentrations of PNGase F for 2 h. A rabbit polyclonal antibody against JAM-A (Zymed) was used for the detection of JAM-A. The asterisk represents N-glycosylated, the open circle N-declycosylated JAM-A. Representative experiments out of at least 5 independent experiments with different EC preparations for each subpanel of the figure are shown.

Techniques Used: Generated, Lysis, Western Blot, Staining

Related Articles

Centrifugation:

Article Title: The small GTPase ARL2 is required for cytokinesis in Trypanosoma brucei
Article Snippet: .. Following centrifugation as above, cells were resuspended in PEME (100 mM PIPES, 2 mM EGTA, 0.1 mM EDTA and 1 mM MgSO4, pH 6.9) containing 1% Nonidet P40, 1× Complete protease inhibitor cocktail (Roche), 7.5 μM Pepstatin A and 5 μM E-64d. .. Pellets were either washed twice in PEME and resuspended in Laemmli buffer (cytoskeleton fraction) or further extracted in PEME containing 1 M NaCl, 200 μg/ml DNaseI, 50 μg/ml RNaseA and protease inhibitors as above.

Article Title: Lys-110 is essential for targeting PCNA to replication and repair foci, and the K110A mutant activates apoptosis
Article Snippet: Briefly, nuclei were lysed in lysis buffer [15 mM Tris/HCl (pH 7.4), 60 mM KCl, 15 mM MgCl2 , 15 mM NaCl, 1mM CaCl2 , 1 mM PMSF, 0.6% Nonidet P40 and 1×protease inhibitor cocktail (Roche)]. .. After centrifugation at 950 g in a bench-top centrifuge (Eppendorf) for 3 min, the pellet was incubated with 20 units of micrococcal nuclease (USB) at 37°C for 10 min.

Stable Transfection:

Article Title: The small GTPase ARL2 is required for cytokinesis in Trypanosoma brucei
Article Snippet: Briefly, parasites stably transfected with pARL2MYC were grown in the presence or absence of tetracycline for 16 h, centrifuged at 800 × g for 10 min at 20 °C, then washed in PBS. .. Following centrifugation as above, cells were resuspended in PEME (100 mM PIPES, 2 mM EGTA, 0.1 mM EDTA and 1 mM MgSO4, pH 6.9) containing 1% Nonidet P40, 1× Complete protease inhibitor cocktail (Roche), 7.5 μM Pepstatin A and 5 μM E-64d.

Construct:

Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
Article Snippet: .. To this end, HEK293T cells, transiently expressing the SF-TAP-tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails II and III (Sigma-Aldrich) in TBS (30 mM Tris-HCl (pH 7.4), 150 mM NaCl), for 20 min at 4 °C. .. After sedimentation of nuclei at 10,000g for 10 min, the protein concentration of the cleared lysates was determined by Bradford before equal protein amounts were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 h. The resin was washed three times with wash buffer (TBS containing 0.1% NP-40, phosphatase inhibitor cocktail II and III).

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Article Snippet: .. HEK293T cells transiently expressing the SF-TAP tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktails I and II (Sigma-Aldrich) in TBS (30 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 20 minutes at 4°C. .. After sedimentation of nuclei at 10,000 g for 10 minutes, the cleared lysates were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 hour before the resin was washed 3 times with wash buffer (TBS containing 0.1% NP-40 and phosphatase inhibitor cocktails I and II).

Incubation:

Article Title: The small GTPase ARL2 is required for cytokinesis in Trypanosoma brucei
Article Snippet: Following centrifugation as above, cells were resuspended in PEME (100 mM PIPES, 2 mM EGTA, 0.1 mM EDTA and 1 mM MgSO4, pH 6.9) containing 1% Nonidet P40, 1× Complete protease inhibitor cocktail (Roche), 7.5 μM Pepstatin A and 5 μM E-64d. .. Samples were incubated on ice for 10 min, centrifuged as above, salt extraction repeated once, then pellets washed twice in PEME.

Article Title: Lys-110 is essential for targeting PCNA to replication and repair foci, and the K110A mutant activates apoptosis
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Article Title: The vesicular nucleotide transporter (VNUT) is involved in the extracellular ATP effect on neuronal differentiation
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Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
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Article Title: Transcript Profiling of Elf5+/− Mammary Glands during Pregnancy Identifies Novel Targets of Elf5
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Article Title: Evaluation of Soluble Junctional Adhesion Molecule-A as a Biomarker of Human Brain Endothelial Barrier Breakdown
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Article Title: Co-operative Cdc42 and Rho signalling mediates ephrinB-triggered endothelial cell retraction
Article Snippet: Briefly, following biotinylation of cell surfaces at 4 °C for 10 min using EZ-Link NHS ( N -hydroxysuccinimido)-SS-biotin (Pierce), cells were maintained at 4 °C, and incubated in a Tris/amine buffer (25 mM Tris/HCl, 137 mM NaCl, 5 mM KCl, 2.3 mM CaCl2 , 0.5 mM MgCl2 and 0.143 g/l Na2 HPO4 , pH 7.4) for 10 min. .. Cells were washed several times in TBS (Tris-buffered saline: 20 mM Tris/HCl and 150 mM NaCl, pH 7.4) before lysates were prepared in a modified RIPA buffer [125 mM NaCl, 20 mM Tris/HCl, 10% (v/v) glycerol, 1% (v/v) Nonidet P40, 50 mM NaF, 200 mM vanadate, 100 μg/ml PMSF and Complete™ protease inhibitor cocktail (Roche)].

Expressing:

Article Title: RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells
Article Snippet: Western blot analysis The expression of c-MYC, cyclin-dependent kinase (CDK) inhibitor p21 (waf1/Cip1) , caspase-9 and β-actin protein was assessed by Western blot analysis. .. In brief, human MB cells transfected with siRNAs for 72 h or treated with chemotherapy for 72 h after 48 h of transfection with siRNAs, were lysed with lysis buffer (1 ml/107 cells, 50 mM Tris-HCl buffer [pH 8.0], 150 mM NaCl, 1% Nonidet P40, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (Complete, Roche; Basel, Switzerland) and incubated on ice for 30 min. After measuring the protein concentration by the BCA method (Pierce; Rockford, USA), 12 μg total protein lysates were separated by 10% SDS-polyacrylamide gels and the gels were subjected to immunoblotting.

Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
Article Snippet: .. To this end, HEK293T cells, transiently expressing the SF-TAP-tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails II and III (Sigma-Aldrich) in TBS (30 mM Tris-HCl (pH 7.4), 150 mM NaCl), for 20 min at 4 °C. .. After sedimentation of nuclei at 10,000g for 10 min, the protein concentration of the cleared lysates was determined by Bradford before equal protein amounts were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 h. The resin was washed three times with wash buffer (TBS containing 0.1% NP-40, phosphatase inhibitor cocktail II and III).

Article Title: The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking
Article Snippet: .. HEK293T cells transiently expressing the SF-TAP tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktails I and II (Sigma-Aldrich) in TBS (30 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 20 minutes at 4°C. .. After sedimentation of nuclei at 10,000 g for 10 minutes, the cleared lysates were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 hour before the resin was washed 3 times with wash buffer (TBS containing 0.1% NP-40 and phosphatase inhibitor cocktails I and II).

BIA-KA:

Article Title: Tumour-initiating cell-specific miR-1246 and miR-1290 expression converge to promote non-small cell lung cancer progression
Article Snippet: Western blot Cells were collected and lysed with Nonidet-P40 supplemented with protease inhibitor cocktail (Roche). .. Western blot Cells were collected and lysed with Nonidet-P40 supplemented with protease inhibitor cocktail (Roche).

Article Title: Induction of Indoleamine 2, 3-Dioxygenase in Human Dendritic Cells by a Cholera Toxin B Subunit—Proinsulin Vaccine
Article Snippet: Vaccinated dendritic cell sample preparation and mass spectrometric analysis After treatment with 10 μg/ml of CTB-INS, the dendritic cell pellet was lysed on ice for 2 hours in radio-immunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, CA) containing 1% Nonidet P40, PMSF (0.2 mM), and protease inhibitor cocktail (Roche). .. Vaccinated dendritic cell sample preparation and mass spectrometric analysis After treatment with 10 μg/ml of CTB-INS, the dendritic cell pellet was lysed on ice for 2 hours in radio-immunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, CA) containing 1% Nonidet P40, PMSF (0.2 mM), and protease inhibitor cocktail (Roche).

Article Title: RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells
Article Snippet: .. In brief, human MB cells transfected with siRNAs for 72 h or treated with chemotherapy for 72 h after 48 h of transfection with siRNAs, were lysed with lysis buffer (1 ml/107 cells, 50 mM Tris-HCl buffer [pH 8.0], 150 mM NaCl, 1% Nonidet P40, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (Complete, Roche; Basel, Switzerland) and incubated on ice for 30 min. After measuring the protein concentration by the BCA method (Pierce; Rockford, USA), 12 μg total protein lysates were separated by 10% SDS-polyacrylamide gels and the gels were subjected to immunoblotting. .. Nonspecific binding sites were blocked by 3 h incubation in TBST (10 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween-20) supplemented with 5% non-fat dry milk.

Modification:

Article Title: Co-operative Cdc42 and Rho signalling mediates ephrinB-triggered endothelial cell retraction
Article Snippet: .. Cells were washed several times in TBS (Tris-buffered saline: 20 mM Tris/HCl and 150 mM NaCl, pH 7.4) before lysates were prepared in a modified RIPA buffer [125 mM NaCl, 20 mM Tris/HCl, 10% (v/v) glycerol, 1% (v/v) Nonidet P40, 50 mM NaF, 200 mM vanadate, 100 μg/ml PMSF and Complete™ protease inhibitor cocktail (Roche)]. .. Biotinylated proteins were precipitated using streptavidin-conjugated agarose beads (Upstate Biotechnology) and were resuspended in Laemmli loading buffer and analysed by SDS/PAGE (?

Western Blot:

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
Article Snippet: .. Western blot analysis Total protein was isolated by lysing cells in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl 1.0% (v/v) nonidet-P40, 0.5% m/v sodium deoxycholate, 0.1% m/v sodium dodecyl sulfate) supplemented with protease inhibitor (Roche). .. Western blot analysis was run with NuPAGE SDS-PAGE gel system (Life Technologies) and primary antibodies against DGKA (11547-1-AP, Proteintech), beta actin (ACTB; sc-47778 HRP, Santa Cruz Biotechnology), PRKCA (2056P, Cell Signaling Technology), collagen 1 (EPR7785, Abcam) and appropriate secondary antibodies (Santa Cruz Biotechnology).

Article Title: Tumour-initiating cell-specific miR-1246 and miR-1290 expression converge to promote non-small cell lung cancer progression
Article Snippet: .. Western blot Cells were collected and lysed with Nonidet-P40 supplemented with protease inhibitor cocktail (Roche). .. Protein concentrations of the extracts were measured using BCA assay (Pierce) and equalized with the extraction reagent.

Article Title: The vesicular nucleotide transporter (VNUT) is involved in the extracellular ATP effect on neuronal differentiation
Article Snippet: .. Western blotting N2a cells were lysed and homogenized for 1 h at 4 °C in lysis buffer containing 50 mM Tris/ HCl, 150 mM NaCl, 1 % Nonidet P40 and CompleteTM Protease Inhibitor Cocktail Tablets (Roche Diagnostics GmbH), pH 7.4. ..

Article Title: RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells
Article Snippet: Paragraph title: Western blot analysis ... In brief, human MB cells transfected with siRNAs for 72 h or treated with chemotherapy for 72 h after 48 h of transfection with siRNAs, were lysed with lysis buffer (1 ml/107 cells, 50 mM Tris-HCl buffer [pH 8.0], 150 mM NaCl, 1% Nonidet P40, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (Complete, Roche; Basel, Switzerland) and incubated on ice for 30 min. After measuring the protein concentration by the BCA method (Pierce; Rockford, USA), 12 μg total protein lysates were separated by 10% SDS-polyacrylamide gels and the gels were subjected to immunoblotting.

Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells
Article Snippet: Paragraph title: Western blotting ... Cells were washed in PBS and then lysed on ice in a lysis buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Nonidet P40 and the Com plete™ protease-inhibitor cocktail (Roche).

Article Title: Evaluation of Soluble Junctional Adhesion Molecule-A as a Biomarker of Human Brain Endothelial Barrier Breakdown
Article Snippet: .. Western Blotting For generation of whole cell protein extracts, cells grown to subconfluency in 25 cm2 flasks were stimulated as indicated, washed with icecold PBS and scraped into radioimmunoprecipitation assay (RIPA) buffer composed of 50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 1% Nonidet P40; 0.25% sodium deoxycholate and 1× Roche COMPLETE® protease inhibitor mix (Roche Diagnostics GmbH, Mannheim, Germany). ..

Transfection:

Article Title: The small GTPase ARL2 is required for cytokinesis in Trypanosoma brucei
Article Snippet: Briefly, parasites stably transfected with pARL2MYC were grown in the presence or absence of tetracycline for 16 h, centrifuged at 800 × g for 10 min at 20 °C, then washed in PBS. .. Following centrifugation as above, cells were resuspended in PEME (100 mM PIPES, 2 mM EGTA, 0.1 mM EDTA and 1 mM MgSO4, pH 6.9) containing 1% Nonidet P40, 1× Complete protease inhibitor cocktail (Roche), 7.5 μM Pepstatin A and 5 μM E-64d.

Article Title: RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells
Article Snippet: .. In brief, human MB cells transfected with siRNAs for 72 h or treated with chemotherapy for 72 h after 48 h of transfection with siRNAs, were lysed with lysis buffer (1 ml/107 cells, 50 mM Tris-HCl buffer [pH 8.0], 150 mM NaCl, 1% Nonidet P40, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (Complete, Roche; Basel, Switzerland) and incubated on ice for 30 min. After measuring the protein concentration by the BCA method (Pierce; Rockford, USA), 12 μg total protein lysates were separated by 10% SDS-polyacrylamide gels and the gels were subjected to immunoblotting. .. Nonspecific binding sites were blocked by 3 h incubation in TBST (10 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween-20) supplemented with 5% non-fat dry milk.

Protease Inhibitor:

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
Article Snippet: .. Western blot analysis Total protein was isolated by lysing cells in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl 1.0% (v/v) nonidet-P40, 0.5% m/v sodium deoxycholate, 0.1% m/v sodium dodecyl sulfate) supplemented with protease inhibitor (Roche). .. Western blot analysis was run with NuPAGE SDS-PAGE gel system (Life Technologies) and primary antibodies against DGKA (11547-1-AP, Proteintech), beta actin (ACTB; sc-47778 HRP, Santa Cruz Biotechnology), PRKCA (2056P, Cell Signaling Technology), collagen 1 (EPR7785, Abcam) and appropriate secondary antibodies (Santa Cruz Biotechnology).

Article Title: The small GTPase ARL2 is required for cytokinesis in Trypanosoma brucei
Article Snippet: .. Following centrifugation as above, cells were resuspended in PEME (100 mM PIPES, 2 mM EGTA, 0.1 mM EDTA and 1 mM MgSO4, pH 6.9) containing 1% Nonidet P40, 1× Complete protease inhibitor cocktail (Roche), 7.5 μM Pepstatin A and 5 μM E-64d. .. Pellets were either washed twice in PEME and resuspended in Laemmli buffer (cytoskeleton fraction) or further extracted in PEME containing 1 M NaCl, 200 μg/ml DNaseI, 50 μg/ml RNaseA and protease inhibitors as above.

Article Title: Tumour-initiating cell-specific miR-1246 and miR-1290 expression converge to promote non-small cell lung cancer progression
Article Snippet: .. Western blot Cells were collected and lysed with Nonidet-P40 supplemented with protease inhibitor cocktail (Roche). .. Protein concentrations of the extracts were measured using BCA assay (Pierce) and equalized with the extraction reagent.

Article Title: Induction of Indoleamine 2, 3-Dioxygenase in Human Dendritic Cells by a Cholera Toxin B Subunit—Proinsulin Vaccine
Article Snippet: .. Vaccinated dendritic cell sample preparation and mass spectrometric analysis After treatment with 10 μg/ml of CTB-INS, the dendritic cell pellet was lysed on ice for 2 hours in radio-immunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, CA) containing 1% Nonidet P40, PMSF (0.2 mM), and protease inhibitor cocktail (Roche). ..

Article Title: PA28 modulates antigen processing and viral replication during coxsackievirus B3 infection
Article Snippet: .. Protein isolation and immunoblot analysis For SDS-PAGE cell or tissue lysis was performed using RIPA buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% (v/v) Nonidet P40, 0.1% (w/v) SDS, 10 μM MG132, 5 mM NEM, Complete® protease inhibitor cocktail (Roche)). .. For native-PAGE, cells were lysed with TSDG buffer (10 mM Tris-HCl pH 7.0, 25 mM KCl, 10 mM NaCl, 1.1 mM MgCl2 , 1 mM DTT, 2 mM ATP, 10% (v/v) glycerol) followed by 4 cycles of snap freezing and thawing.

Article Title: The vesicular nucleotide transporter (VNUT) is involved in the extracellular ATP effect on neuronal differentiation
Article Snippet: .. Western blotting N2a cells were lysed and homogenized for 1 h at 4 °C in lysis buffer containing 50 mM Tris/ HCl, 150 mM NaCl, 1 % Nonidet P40 and CompleteTM Protease Inhibitor Cocktail Tablets (Roche Diagnostics GmbH), pH 7.4. ..

Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
Article Snippet: .. To this end, HEK293T cells, transiently expressing the SF-TAP-tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails II and III (Sigma-Aldrich) in TBS (30 mM Tris-HCl (pH 7.4), 150 mM NaCl), for 20 min at 4 °C. .. After sedimentation of nuclei at 10,000g for 10 min, the protein concentration of the cleared lysates was determined by Bradford before equal protein amounts were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 h. The resin was washed three times with wash buffer (TBS containing 0.1% NP-40, phosphatase inhibitor cocktail II and III).

Article Title: The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking
Article Snippet: .. HEK293T cells transiently expressing the SF-TAP tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktails I and II (Sigma-Aldrich) in TBS (30 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 20 minutes at 4°C. .. After sedimentation of nuclei at 10,000 g for 10 minutes, the cleared lysates were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 hour before the resin was washed 3 times with wash buffer (TBS containing 0.1% NP-40 and phosphatase inhibitor cocktails I and II).

Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells
Article Snippet: .. Cells were washed in PBS and then lysed on ice in a lysis buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Nonidet P40 and the Com plete™ protease-inhibitor cocktail (Roche). .. Protein concentration was determined using the bicinchoninic acid reagent (Pierce Chemical Company, Rockford, IL, U.S.A.).

Article Title: Evaluation of Soluble Junctional Adhesion Molecule-A as a Biomarker of Human Brain Endothelial Barrier Breakdown
Article Snippet: .. Western Blotting For generation of whole cell protein extracts, cells grown to subconfluency in 25 cm2 flasks were stimulated as indicated, washed with icecold PBS and scraped into radioimmunoprecipitation assay (RIPA) buffer composed of 50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 1% Nonidet P40; 0.25% sodium deoxycholate and 1× Roche COMPLETE® protease inhibitor mix (Roche Diagnostics GmbH, Mannheim, Germany). ..

Article Title: Co-operative Cdc42 and Rho signalling mediates ephrinB-triggered endothelial cell retraction
Article Snippet: .. Cells were washed several times in TBS (Tris-buffered saline: 20 mM Tris/HCl and 150 mM NaCl, pH 7.4) before lysates were prepared in a modified RIPA buffer [125 mM NaCl, 20 mM Tris/HCl, 10% (v/v) glycerol, 1% (v/v) Nonidet P40, 50 mM NaF, 200 mM vanadate, 100 μg/ml PMSF and Complete™ protease inhibitor cocktail (Roche)]. .. Biotinylated proteins were precipitated using streptavidin-conjugated agarose beads (Upstate Biotechnology) and were resuspended in Laemmli loading buffer and analysed by SDS/PAGE (?

Cell Culture:

Article Title: Evaluation of Soluble Junctional Adhesion Molecule-A as a Biomarker of Human Brain Endothelial Barrier Breakdown
Article Snippet: Western Blotting For generation of whole cell protein extracts, cells grown to subconfluency in 25 cm2 flasks were stimulated as indicated, washed with icecold PBS and scraped into radioimmunoprecipitation assay (RIPA) buffer composed of 50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 1% Nonidet P40; 0.25% sodium deoxycholate and 1× Roche COMPLETE® protease inhibitor mix (Roche Diagnostics GmbH, Mannheim, Germany). .. For analysis of sJAM-A levels in cell culture supernatants 1.3 mL cell culture supernatant were transferred to an 1.5 mL Eppendorf tube.

Sedimentation:

Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
Article Snippet: To this end, HEK293T cells, transiently expressing the SF-TAP-tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails II and III (Sigma-Aldrich) in TBS (30 mM Tris-HCl (pH 7.4), 150 mM NaCl), for 20 min at 4 °C. .. After sedimentation of nuclei at 10,000g for 10 min, the protein concentration of the cleared lysates was determined by Bradford before equal protein amounts were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 h. The resin was washed three times with wash buffer (TBS containing 0.1% NP-40, phosphatase inhibitor cocktail II and III).

Article Title: The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking
Article Snippet: HEK293T cells transiently expressing the SF-TAP tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktails I and II (Sigma-Aldrich) in TBS (30 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 20 minutes at 4°C. .. After sedimentation of nuclei at 10,000 g for 10 minutes, the cleared lysates were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 hour before the resin was washed 3 times with wash buffer (TBS containing 0.1% NP-40 and phosphatase inhibitor cocktails I and II).

other:

Article Title: MT6-MMP is present in lipid rafts and faces inward in living human PMNs but translocates to the cell surface during neutrophil apoptosis
Article Snippet: Triton X-100 and Nonidet P40 were from Roche (Laval, Quebec, Canada).

Protein Concentration:

Article Title: Induction of Indoleamine 2, 3-Dioxygenase in Human Dendritic Cells by a Cholera Toxin B Subunit—Proinsulin Vaccine
Article Snippet: Vaccinated dendritic cell sample preparation and mass spectrometric analysis After treatment with 10 μg/ml of CTB-INS, the dendritic cell pellet was lysed on ice for 2 hours in radio-immunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, CA) containing 1% Nonidet P40, PMSF (0.2 mM), and protease inhibitor cocktail (Roche). .. Vaccinated dendritic cell sample preparation and mass spectrometric analysis After treatment with 10 μg/ml of CTB-INS, the dendritic cell pellet was lysed on ice for 2 hours in radio-immunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, CA) containing 1% Nonidet P40, PMSF (0.2 mM), and protease inhibitor cocktail (Roche).

Article Title: RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells
Article Snippet: .. In brief, human MB cells transfected with siRNAs for 72 h or treated with chemotherapy for 72 h after 48 h of transfection with siRNAs, were lysed with lysis buffer (1 ml/107 cells, 50 mM Tris-HCl buffer [pH 8.0], 150 mM NaCl, 1% Nonidet P40, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (Complete, Roche; Basel, Switzerland) and incubated on ice for 30 min. After measuring the protein concentration by the BCA method (Pierce; Rockford, USA), 12 μg total protein lysates were separated by 10% SDS-polyacrylamide gels and the gels were subjected to immunoblotting. .. Nonspecific binding sites were blocked by 3 h incubation in TBST (10 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween-20) supplemented with 5% non-fat dry milk.

Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
Article Snippet: To this end, HEK293T cells, transiently expressing the SF-TAP-tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails II and III (Sigma-Aldrich) in TBS (30 mM Tris-HCl (pH 7.4), 150 mM NaCl), for 20 min at 4 °C. .. After sedimentation of nuclei at 10,000g for 10 min, the protein concentration of the cleared lysates was determined by Bradford before equal protein amounts were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 h. The resin was washed three times with wash buffer (TBS containing 0.1% NP-40, phosphatase inhibitor cocktail II and III).

Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells
Article Snippet: Cells were washed in PBS and then lysed on ice in a lysis buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Nonidet P40 and the Com plete™ protease-inhibitor cocktail (Roche). .. Protein concentration was determined using the bicinchoninic acid reagent (Pierce Chemical Company, Rockford, IL, U.S.A.).

Affinity Purification:

Article Title: The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking
Article Snippet: Paragraph title: Affinity purification of protein complexes ... HEK293T cells transiently expressing the SF-TAP tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktails I and II (Sigma-Aldrich) in TBS (30 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 20 minutes at 4°C.

Binding Assay:

Article Title: RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells
Article Snippet: In brief, human MB cells transfected with siRNAs for 72 h or treated with chemotherapy for 72 h after 48 h of transfection with siRNAs, were lysed with lysis buffer (1 ml/107 cells, 50 mM Tris-HCl buffer [pH 8.0], 150 mM NaCl, 1% Nonidet P40, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (Complete, Roche; Basel, Switzerland) and incubated on ice for 30 min. After measuring the protein concentration by the BCA method (Pierce; Rockford, USA), 12 μg total protein lysates were separated by 10% SDS-polyacrylamide gels and the gels were subjected to immunoblotting. .. Nonspecific binding sites were blocked by 3 h incubation in TBST (10 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween-20) supplemented with 5% non-fat dry milk.

Isolation:

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
Article Snippet: .. Western blot analysis Total protein was isolated by lysing cells in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl 1.0% (v/v) nonidet-P40, 0.5% m/v sodium deoxycholate, 0.1% m/v sodium dodecyl sulfate) supplemented with protease inhibitor (Roche). .. Western blot analysis was run with NuPAGE SDS-PAGE gel system (Life Technologies) and primary antibodies against DGKA (11547-1-AP, Proteintech), beta actin (ACTB; sc-47778 HRP, Santa Cruz Biotechnology), PRKCA (2056P, Cell Signaling Technology), collagen 1 (EPR7785, Abcam) and appropriate secondary antibodies (Santa Cruz Biotechnology).

Article Title: PA28 modulates antigen processing and viral replication during coxsackievirus B3 infection
Article Snippet: .. Protein isolation and immunoblot analysis For SDS-PAGE cell or tissue lysis was performed using RIPA buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% (v/v) Nonidet P40, 0.1% (w/v) SDS, 10 μM MG132, 5 mM NEM, Complete® protease inhibitor cocktail (Roche)). .. For native-PAGE, cells were lysed with TSDG buffer (10 mM Tris-HCl pH 7.0, 25 mM KCl, 10 mM NaCl, 1.1 mM MgCl2 , 1 mM DTT, 2 mM ATP, 10% (v/v) glycerol) followed by 4 cycles of snap freezing and thawing.

Purification:

Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
Article Snippet: SF-TAP-tagged proteins and associated protein complexes were purified from HEK293T cells . .. To this end, HEK293T cells, transiently expressing the SF-TAP-tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails II and III (Sigma-Aldrich) in TBS (30 mM Tris-HCl (pH 7.4), 150 mM NaCl), for 20 min at 4 °C.

Article Title: The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking
Article Snippet: For one-step Strep purifications, SF-TAP–tagged proteins and associated protein complexes were purified essentially as described previously [ , ]. .. HEK293T cells transiently expressing the SF-TAP tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktails I and II (Sigma-Aldrich) in TBS (30 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 20 minutes at 4°C.

Polyacrylamide Gel Electrophoresis:

Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells
Article Snippet: Cells were washed in PBS and then lysed on ice in a lysis buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Nonidet P40 and the Com plete™ protease-inhibitor cocktail (Roche). .. The lysates (50 μg of protein) were separated on SDS/12.5% (w/v) PAGE gels and electroblotted on to a nitrocellulose membrane.

SDS Page:

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
Article Snippet: Western blot analysis Total protein was isolated by lysing cells in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl 1.0% (v/v) nonidet-P40, 0.5% m/v sodium deoxycholate, 0.1% m/v sodium dodecyl sulfate) supplemented with protease inhibitor (Roche). .. Western blot analysis was run with NuPAGE SDS-PAGE gel system (Life Technologies) and primary antibodies against DGKA (11547-1-AP, Proteintech), beta actin (ACTB; sc-47778 HRP, Santa Cruz Biotechnology), PRKCA (2056P, Cell Signaling Technology), collagen 1 (EPR7785, Abcam) and appropriate secondary antibodies (Santa Cruz Biotechnology).

Article Title: Tumour-initiating cell-specific miR-1246 and miR-1290 expression converge to promote non-small cell lung cancer progression
Article Snippet: Western blot Cells were collected and lysed with Nonidet-P40 supplemented with protease inhibitor cocktail (Roche). .. Equal amount of the extracts was loaded and subjected to SDS-PAGE, transferred onto nitrocellulose membranes.

Article Title: PA28 modulates antigen processing and viral replication during coxsackievirus B3 infection
Article Snippet: .. Protein isolation and immunoblot analysis For SDS-PAGE cell or tissue lysis was performed using RIPA buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% (v/v) Nonidet P40, 0.1% (w/v) SDS, 10 μM MG132, 5 mM NEM, Complete® protease inhibitor cocktail (Roche)). .. For native-PAGE, cells were lysed with TSDG buffer (10 mM Tris-HCl pH 7.0, 25 mM KCl, 10 mM NaCl, 1.1 mM MgCl2 , 1 mM DTT, 2 mM ATP, 10% (v/v) glycerol) followed by 4 cycles of snap freezing and thawing.

Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
Article Snippet: To this end, HEK293T cells, transiently expressing the SF-TAP-tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails II and III (Sigma-Aldrich) in TBS (30 mM Tris-HCl (pH 7.4), 150 mM NaCl), for 20 min at 4 °C. .. The eluted samples were combined before concentration using 10 kDa cut-off VivaSpin 500 centrifugal devices (Sartorius Stedim Biotech) and pre-fractionation using SDS–PAGE and in-gel tryptic cleavage .

Article Title: Co-operative Cdc42 and Rho signalling mediates ephrinB-triggered endothelial cell retraction
Article Snippet: Cells were washed several times in TBS (Tris-buffered saline: 20 mM Tris/HCl and 150 mM NaCl, pH 7.4) before lysates were prepared in a modified RIPA buffer [125 mM NaCl, 20 mM Tris/HCl, 10% (v/v) glycerol, 1% (v/v) Nonidet P40, 50 mM NaF, 200 mM vanadate, 100 μg/ml PMSF and Complete™ protease inhibitor cocktail (Roche)]. .. Biotinylated proteins were precipitated using streptavidin-conjugated agarose beads (Upstate Biotechnology) and were resuspended in Laemmli loading buffer and analysed by SDS/PAGE (?

Software:

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
Article Snippet: Western blot analysis Total protein was isolated by lysing cells in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl 1.0% (v/v) nonidet-P40, 0.5% m/v sodium deoxycholate, 0.1% m/v sodium dodecyl sulfate) supplemented with protease inhibitor (Roche). .. Band intensities were quantitated with ImageJ software using beta actin band intensities for normalization.

Sample Prep:

Article Title: Induction of Indoleamine 2, 3-Dioxygenase in Human Dendritic Cells by a Cholera Toxin B Subunit—Proinsulin Vaccine
Article Snippet: .. Vaccinated dendritic cell sample preparation and mass spectrometric analysis After treatment with 10 μg/ml of CTB-INS, the dendritic cell pellet was lysed on ice for 2 hours in radio-immunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, CA) containing 1% Nonidet P40, PMSF (0.2 mM), and protease inhibitor cocktail (Roche). ..

Radio Immunoprecipitation:

Article Title: Induction of Indoleamine 2, 3-Dioxygenase in Human Dendritic Cells by a Cholera Toxin B Subunit—Proinsulin Vaccine
Article Snippet: .. Vaccinated dendritic cell sample preparation and mass spectrometric analysis After treatment with 10 μg/ml of CTB-INS, the dendritic cell pellet was lysed on ice for 2 hours in radio-immunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, CA) containing 1% Nonidet P40, PMSF (0.2 mM), and protease inhibitor cocktail (Roche). ..

Article Title: Evaluation of Soluble Junctional Adhesion Molecule-A as a Biomarker of Human Brain Endothelial Barrier Breakdown
Article Snippet: .. Western Blotting For generation of whole cell protein extracts, cells grown to subconfluency in 25 cm2 flasks were stimulated as indicated, washed with icecold PBS and scraped into radioimmunoprecipitation assay (RIPA) buffer composed of 50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 1% Nonidet P40; 0.25% sodium deoxycholate and 1× Roche COMPLETE® protease inhibitor mix (Roche Diagnostics GmbH, Mannheim, Germany). ..

CtB Assay:

Article Title: Induction of Indoleamine 2, 3-Dioxygenase in Human Dendritic Cells by a Cholera Toxin B Subunit—Proinsulin Vaccine
Article Snippet: .. Vaccinated dendritic cell sample preparation and mass spectrometric analysis After treatment with 10 μg/ml of CTB-INS, the dendritic cell pellet was lysed on ice for 2 hours in radio-immunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, CA) containing 1% Nonidet P40, PMSF (0.2 mM), and protease inhibitor cocktail (Roche). ..

Concentration Assay:

Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
Article Snippet: To this end, HEK293T cells, transiently expressing the SF-TAP-tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails II and III (Sigma-Aldrich) in TBS (30 mM Tris-HCl (pH 7.4), 150 mM NaCl), for 20 min at 4 °C. .. The eluted samples were combined before concentration using 10 kDa cut-off VivaSpin 500 centrifugal devices (Sartorius Stedim Biotech) and pre-fractionation using SDS–PAGE and in-gel tryptic cleavage .

Fractionation:

Article Title: The small GTPase ARL2 is required for cytokinesis in Trypanosoma brucei
Article Snippet: Paragraph title: Subcellular fractionation ... Following centrifugation as above, cells were resuspended in PEME (100 mM PIPES, 2 mM EGTA, 0.1 mM EDTA and 1 mM MgSO4, pH 6.9) containing 1% Nonidet P40, 1× Complete protease inhibitor cocktail (Roche), 7.5 μM Pepstatin A and 5 μM E-64d.

Article Title: Lys-110 is essential for targeting PCNA to replication and repair foci, and the K110A mutant activates apoptosis
Article Snippet: The preparation of nuclear fractionation and chromatin-binding assays were as described previously ( ; ; ). .. Briefly, nuclei were lysed in lysis buffer [15 mM Tris/HCl (pH 7.4), 60 mM KCl, 15 mM MgCl2 , 15 mM NaCl, 1mM CaCl2 , 1 mM PMSF, 0.6% Nonidet P40 and 1×protease inhibitor cocktail (Roche)].

Lysis:

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
Article Snippet: .. Western blot analysis Total protein was isolated by lysing cells in RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl 1.0% (v/v) nonidet-P40, 0.5% m/v sodium deoxycholate, 0.1% m/v sodium dodecyl sulfate) supplemented with protease inhibitor (Roche). .. Western blot analysis was run with NuPAGE SDS-PAGE gel system (Life Technologies) and primary antibodies against DGKA (11547-1-AP, Proteintech), beta actin (ACTB; sc-47778 HRP, Santa Cruz Biotechnology), PRKCA (2056P, Cell Signaling Technology), collagen 1 (EPR7785, Abcam) and appropriate secondary antibodies (Santa Cruz Biotechnology).

Article Title: Lys-110 is essential for targeting PCNA to replication and repair foci, and the K110A mutant activates apoptosis
Article Snippet: .. Briefly, nuclei were lysed in lysis buffer [15 mM Tris/HCl (pH 7.4), 60 mM KCl, 15 mM MgCl2 , 15 mM NaCl, 1mM CaCl2 , 1 mM PMSF, 0.6% Nonidet P40 and 1×protease inhibitor cocktail (Roche)]. .. After centrifugation at 950 g in a bench-top centrifuge (Eppendorf) for 3 min, the pellet was incubated with 20 units of micrococcal nuclease (USB) at 37°C for 10 min.

Article Title: PA28 modulates antigen processing and viral replication during coxsackievirus B3 infection
Article Snippet: .. Protein isolation and immunoblot analysis For SDS-PAGE cell or tissue lysis was performed using RIPA buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% (v/v) Nonidet P40, 0.1% (w/v) SDS, 10 μM MG132, 5 mM NEM, Complete® protease inhibitor cocktail (Roche)). .. For native-PAGE, cells were lysed with TSDG buffer (10 mM Tris-HCl pH 7.0, 25 mM KCl, 10 mM NaCl, 1.1 mM MgCl2 , 1 mM DTT, 2 mM ATP, 10% (v/v) glycerol) followed by 4 cycles of snap freezing and thawing.

Article Title: The vesicular nucleotide transporter (VNUT) is involved in the extracellular ATP effect on neuronal differentiation
Article Snippet: .. Western blotting N2a cells were lysed and homogenized for 1 h at 4 °C in lysis buffer containing 50 mM Tris/ HCl, 150 mM NaCl, 1 % Nonidet P40 and CompleteTM Protease Inhibitor Cocktail Tablets (Roche Diagnostics GmbH), pH 7.4. ..

Article Title: RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells
Article Snippet: .. In brief, human MB cells transfected with siRNAs for 72 h or treated with chemotherapy for 72 h after 48 h of transfection with siRNAs, were lysed with lysis buffer (1 ml/107 cells, 50 mM Tris-HCl buffer [pH 8.0], 150 mM NaCl, 1% Nonidet P40, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (Complete, Roche; Basel, Switzerland) and incubated on ice for 30 min. After measuring the protein concentration by the BCA method (Pierce; Rockford, USA), 12 μg total protein lysates were separated by 10% SDS-polyacrylamide gels and the gels were subjected to immunoblotting. .. Nonspecific binding sites were blocked by 3 h incubation in TBST (10 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween-20) supplemented with 5% non-fat dry milk.

Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
Article Snippet: .. To this end, HEK293T cells, transiently expressing the SF-TAP-tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails II and III (Sigma-Aldrich) in TBS (30 mM Tris-HCl (pH 7.4), 150 mM NaCl), for 20 min at 4 °C. .. After sedimentation of nuclei at 10,000g for 10 min, the protein concentration of the cleared lysates was determined by Bradford before equal protein amounts were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 h. The resin was washed three times with wash buffer (TBS containing 0.1% NP-40, phosphatase inhibitor cocktail II and III).

Article Title: The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking
Article Snippet: .. HEK293T cells transiently expressing the SF-TAP tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktails I and II (Sigma-Aldrich) in TBS (30 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 20 minutes at 4°C. .. After sedimentation of nuclei at 10,000 g for 10 minutes, the cleared lysates were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 hour before the resin was washed 3 times with wash buffer (TBS containing 0.1% NP-40 and phosphatase inhibitor cocktails I and II).

Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells
Article Snippet: .. Cells were washed in PBS and then lysed on ice in a lysis buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Nonidet P40 and the Com plete™ protease-inhibitor cocktail (Roche). .. Protein concentration was determined using the bicinchoninic acid reagent (Pierce Chemical Company, Rockford, IL, U.S.A.).

Clear Native PAGE:

Article Title: PA28 modulates antigen processing and viral replication during coxsackievirus B3 infection
Article Snippet: Protein isolation and immunoblot analysis For SDS-PAGE cell or tissue lysis was performed using RIPA buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% (v/v) Nonidet P40, 0.1% (w/v) SDS, 10 μM MG132, 5 mM NEM, Complete® protease inhibitor cocktail (Roche)). .. For native-PAGE, cells were lysed with TSDG buffer (10 mM Tris-HCl pH 7.0, 25 mM KCl, 10 mM NaCl, 1.1 mM MgCl2 , 1 mM DTT, 2 mM ATP, 10% (v/v) glycerol) followed by 4 cycles of snap freezing and thawing.

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  • 99
    Roche ripa buffer
    Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in <t>293T</t> cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with <t>RIPA</t> buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.
    Ripa Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 4719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ripa buffer/product/Roche
    Average 99 stars, based on 4719 article reviews
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    Roche nonidet p40
    Substitution of Lys-110 with an alanine residue results in the accumulation of the mutant PCNA in the heterochromatin region ( A ) The cell fractionation protocol. CHO cells transfected with WT or mutant constructs were fractionated at 12 h post-transfection. MNase, micrococcal nuclease; NP-40, <t>Nonidet</t> <t>P40;</t> Sup, supernatant. ( B ) Fractionated samples were analysed by SDS/PAGE and Western blotting with anti-GFP, -PCNA, -(histone H1) or -HP1α antibodies as indicated. The striking differences in chromatin localization of PCNA(K110A) and endogenous PCNA are shown with * and arrowheads. ( C ) DNA purified from each fraction was separated by agarose gel electrophoresis (2% gel). Di, di-nucleosome; M, DNA size makers; Mono, mononucleosome.
    Nonidet P40, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche nt rna immunoprecipitation buffer
    Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by <t>RNA</t> <t>immunoprecipitation</t> assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008
    Nt Rna Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.

    Journal: Oncotarget

    Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    doi: 10.18632/oncotarget.8072

    Figure Lengend Snippet: Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.

    Article Snippet: After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany).

    Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Staining, Plasmid Preparation, Mutagenesis, Transfection

    Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.

    Journal: Oncotarget

    Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    doi: 10.18632/oncotarget.8072

    Figure Lengend Snippet: Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.

    Article Snippet: After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany).

    Techniques: Incubation, Immunoprecipitation, Transfection, Western Blot

    Substitution of Lys-110 with an alanine residue results in the accumulation of the mutant PCNA in the heterochromatin region ( A ) The cell fractionation protocol. CHO cells transfected with WT or mutant constructs were fractionated at 12 h post-transfection. MNase, micrococcal nuclease; NP-40, Nonidet P40; Sup, supernatant. ( B ) Fractionated samples were analysed by SDS/PAGE and Western blotting with anti-GFP, -PCNA, -(histone H1) or -HP1α antibodies as indicated. The striking differences in chromatin localization of PCNA(K110A) and endogenous PCNA are shown with * and arrowheads. ( C ) DNA purified from each fraction was separated by agarose gel electrophoresis (2% gel). Di, di-nucleosome; M, DNA size makers; Mono, mononucleosome.

    Journal: Biology of the cell

    Article Title: Lys-110 is essential for targeting PCNA to replication and repair foci, and the K110A mutant activates apoptosis

    doi: 10.1042/BC20070158

    Figure Lengend Snippet: Substitution of Lys-110 with an alanine residue results in the accumulation of the mutant PCNA in the heterochromatin region ( A ) The cell fractionation protocol. CHO cells transfected with WT or mutant constructs were fractionated at 12 h post-transfection. MNase, micrococcal nuclease; NP-40, Nonidet P40; Sup, supernatant. ( B ) Fractionated samples were analysed by SDS/PAGE and Western blotting with anti-GFP, -PCNA, -(histone H1) or -HP1α antibodies as indicated. The striking differences in chromatin localization of PCNA(K110A) and endogenous PCNA are shown with * and arrowheads. ( C ) DNA purified from each fraction was separated by agarose gel electrophoresis (2% gel). Di, di-nucleosome; M, DNA size makers; Mono, mononucleosome.

    Article Snippet: Briefly, nuclei were lysed in lysis buffer [15 mM Tris/HCl (pH 7.4), 60 mM KCl, 15 mM MgCl2 , 15 mM NaCl, 1mM CaCl2 , 1 mM PMSF, 0.6% Nonidet P40 and 1×protease inhibitor cocktail (Roche)].

    Techniques: Mutagenesis, Cell Fractionation, Transfection, Construct, SDS Page, Western Blot, Purification, Agarose Gel Electrophoresis

    Detection of intracellular CD83 in monocytes, macrophages and imDCs ( A ) Monocytes (Mo), macrophages (MΦ) and imDCs were fixed with paraformaldehyde and permeabilized with saponin. Upon blocking with 20% (v/v) goat serum, the cells were stained with a PE-labelled CD83 antibody (clone HB15e; filled histograms) or, as a control, isotype IgG (open histograms). The monocytic THP-1 cells were used as controls. ( B ) Monocytes (lane 3), resting macrophages (lane 4), LPS-activated macrophages (lane 5), imDCs (lane 6) and mDCs (lane 7) were lysed with Nonidet P40, and the soluble fractions were analysed by Western blotting using the HB15a CD83 antibody (Santa Cruz Biotechnology). Aliquots (50 μg) of each sample were separated by SDS/PAGE. Also included in the blots was the soluble fraction of THP-1 cells (lane 8), and 293T cells transfected with the pcDNA3.1 plasmid (lane 1) or the phCD83 expression vector (lane 2). The transfected cells were cultured for 24 h. For monocytes, both the soluble (lane 9) and insoluble (lane 10) fractions were subjected to Western blot analysis. ( C ) Expression of CD83 by 293T cells: 293T cells were transfected with the phCD83 expression vector (lower panel) or, as a control, with the pcDNA3.1 plasmid (upper panel). The cells were stained with a PE-labelled CD83 antibody (HB15e; open histograms). The filled histograms represent signals detected with isotype IgG. All results shown are representative of at least three similar experiments.

    Journal: Biochemical Journal

    Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells

    doi: 10.1042/BJ20040741

    Figure Lengend Snippet: Detection of intracellular CD83 in monocytes, macrophages and imDCs ( A ) Monocytes (Mo), macrophages (MΦ) and imDCs were fixed with paraformaldehyde and permeabilized with saponin. Upon blocking with 20% (v/v) goat serum, the cells were stained with a PE-labelled CD83 antibody (clone HB15e; filled histograms) or, as a control, isotype IgG (open histograms). The monocytic THP-1 cells were used as controls. ( B ) Monocytes (lane 3), resting macrophages (lane 4), LPS-activated macrophages (lane 5), imDCs (lane 6) and mDCs (lane 7) were lysed with Nonidet P40, and the soluble fractions were analysed by Western blotting using the HB15a CD83 antibody (Santa Cruz Biotechnology). Aliquots (50 μg) of each sample were separated by SDS/PAGE. Also included in the blots was the soluble fraction of THP-1 cells (lane 8), and 293T cells transfected with the pcDNA3.1 plasmid (lane 1) or the phCD83 expression vector (lane 2). The transfected cells were cultured for 24 h. For monocytes, both the soluble (lane 9) and insoluble (lane 10) fractions were subjected to Western blot analysis. ( C ) Expression of CD83 by 293T cells: 293T cells were transfected with the phCD83 expression vector (lower panel) or, as a control, with the pcDNA3.1 plasmid (upper panel). The cells were stained with a PE-labelled CD83 antibody (HB15e; open histograms). The filled histograms represent signals detected with isotype IgG. All results shown are representative of at least three similar experiments.

    Article Snippet: Cells were washed in PBS and then lysed on ice in a lysis buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Nonidet P40 and the Com plete™ protease-inhibitor cocktail (Roche).

    Techniques: Blocking Assay, Staining, Western Blot, SDS Page, Transfection, Plasmid Preparation, Expressing, Cell Culture

    Surface induction of CD83 on DCs requires Asn-linked glycosylation ( A ) Transfected 293T cells were lysed, and the soluble fraction was either untreated (lane 1) or treated with PNGase F (lane 2). The samples were then separated on SDS/PAGE gels and subjected to Western blotting using the HB15a CD83 antibody. The transfected 293T cells were also cultured in the presence of tunicamycin (lane 3) or DMSO (lane 4) for 24 h, and then lysed with Nonidet P40. The soluble fraction was subjected to SDS/PAGE, followed by Western blotting using the CD83 antibody. ImDCs were activated with LPS in the presence of tunicamycin (lane 5) or DMSO (lane 6) for 6 h, and were similarly analysed by Western blotting using the CD83 antibody. The molecular-mass standards are shown, and apply to all three blots. ( B ) Transfected 293T cells were cultured for 24 h in the presence of tunicamycin (solid line) or DMSO (dotted line), and then stained with the CD83 antibody and analysed by flow cytometry. The filled histogram represents signals on transfected 293T cells cultured in the absence of tunicamycin and DMSO. ( C ) ImDCs were activated with LPS in the presence of tunicamycin (solid line) or DMSO (dotted line), or were unactivated (filled histogram) and then stained with the CD83 antibody and analysed by flow cytometry. The results are representative of two or three independent experiments.

    Journal: Biochemical Journal

    Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells

    doi: 10.1042/BJ20040741

    Figure Lengend Snippet: Surface induction of CD83 on DCs requires Asn-linked glycosylation ( A ) Transfected 293T cells were lysed, and the soluble fraction was either untreated (lane 1) or treated with PNGase F (lane 2). The samples were then separated on SDS/PAGE gels and subjected to Western blotting using the HB15a CD83 antibody. The transfected 293T cells were also cultured in the presence of tunicamycin (lane 3) or DMSO (lane 4) for 24 h, and then lysed with Nonidet P40. The soluble fraction was subjected to SDS/PAGE, followed by Western blotting using the CD83 antibody. ImDCs were activated with LPS in the presence of tunicamycin (lane 5) or DMSO (lane 6) for 6 h, and were similarly analysed by Western blotting using the CD83 antibody. The molecular-mass standards are shown, and apply to all three blots. ( B ) Transfected 293T cells were cultured for 24 h in the presence of tunicamycin (solid line) or DMSO (dotted line), and then stained with the CD83 antibody and analysed by flow cytometry. The filled histogram represents signals on transfected 293T cells cultured in the absence of tunicamycin and DMSO. ( C ) ImDCs were activated with LPS in the presence of tunicamycin (solid line) or DMSO (dotted line), or were unactivated (filled histogram) and then stained with the CD83 antibody and analysed by flow cytometry. The results are representative of two or three independent experiments.

    Article Snippet: Cells were washed in PBS and then lysed on ice in a lysis buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Nonidet P40 and the Com plete™ protease-inhibitor cocktail (Roche).

    Techniques: Transfection, SDS Page, Western Blot, Cell Culture, Staining, Flow Cytometry, Cytometry

    Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by RNA immunoprecipitation assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008

    Journal: eLife

    Article Title: RNA binding protein Caprin-2 is a pivotal regulator of the central osmotic defense response

    doi: 10.7554/eLife.09656

    Figure Lengend Snippet: Caprin-2 binds to the AVP mRNA in the SON and PVN. Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by RNA immunoprecipitation assay. ( A ) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. ( B ) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). ( C ) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05. DOI: http://dx.doi.org/10.7554/eLife.09656.008

    Article Snippet: After homogenization in 100 μl of NT- RNA immunoprecipitation buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2 , 0.5% Nonidet P40, 1 mM EDTA pH 8.0, 1 mM DTT), Complete protease inhibitor (Roche), 200 U/ml RNase Out (Invitrogen, USA) samples were incubated for 10 min on ice and pre-cleared with 25 μl of Protein G-coated Dynabeads (Life Technologies).

    Techniques: Binding Assay, Immunoprecipitation, Incubation, Quantitative RT-PCR