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

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Clone Assay:

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Article Snippet: Clones were washed twice in PBS (with Mg2+ and Ca2+ ) and fixed in 3.7% formaldehyde. .. Cells were permeabilized in 0.2% Nonidet P40 (Roche) and blocked in 10% goat serum.

Centrifugation:

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Article Title: The small GTPase ARL2 is required for cytokinesis in Trypanosoma brucei
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Article Title: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
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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.

Bradford Assay:

Article Title: Autophagy links antimicrobial activity with antigen presentation in Langerhans cells
Article Snippet: LCDC, 1 × 106 per condition, were collected in NP-40 lysis buffer (50 mM Tris [pH 7.4], 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3 VO4 , 1% Nonidet P40 [NP40],0.02% NaN3 and 1 mM PMSF) containing complete protease inhibitors (Roche Applied Science). .. Total protein from cell lysates were quantified using Bradford Assay.

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).

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).

Enzymatic Assay:

Article Title: Sheddase Activity of Tumor Necrosis Factor-Alpha Converting Enzyme is Increased and Prognostically Valuable in Head and Neck Cancer
Article Snippet: Briefly, cultured cells (1 × 106 ) were lysed in 50 μl of lysis buffer, and fresh tissues (50 mg) were homogenized using the tissue grinder system (Fisher Scientific, Pittsburgh, PA) and lysed in 500 μl of lysis buffer on ice, for 1 h. The lysis buffer was composed of Tris-HCl (50 mM), NaCl (150 mM), SDS (0.1%), sodium deoxycholate (1%), Nonidet P40 (1%) and Triton X-100 (1%), and EDTA-free protease inhibitor cocktail (Roche Applied Science). .. TACE enzymatic assay was performed with the 150 μl mixture of 10 μg lysate proteins, assay buffer [Tris-HCl (50 mM, pH 7.4), NaCl (25 mM), glycerol (4%), EDTA-free protease inhibitor cocktail (Roche Applied Science)], and 50 μM of TACE-specific fluorophore/quencher-capped-end substrate with the amino-acid sequence Abz-Leu-Ala-Gln- Ala-Val -Arg-Ser-Ser-Ser-Arg-Dap(Dnp)-NH2 containing the TACE specific cleavage site Ala-Val (Peptide International, Louisville, KY).

Activity Assay:

Article Title: Sheddase Activity of Tumor Necrosis Factor-Alpha Converting Enzyme is Increased and Prognostically Valuable in Head and Neck Cancer
Article Snippet: Paragraph title: TACE sheddase activity assay ... Briefly, cultured cells (1 × 106 ) were lysed in 50 μl of lysis buffer, and fresh tissues (50 mg) were homogenized using the tissue grinder system (Fisher Scientific, Pittsburgh, PA) and lysed in 500 μl of lysis buffer on ice, for 1 h. The lysis buffer was composed of Tris-HCl (50 mM), NaCl (150 mM), SDS (0.1%), sodium deoxycholate (1%), Nonidet P40 (1%) and Triton X-100 (1%), and EDTA-free protease inhibitor cocktail (Roche Applied Science).

Infection:

Article Title: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
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Expressing:

Article Title: RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells
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Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms
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Article Title: The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking
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BIA-KA:

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Article Title: Induction of Indoleamine 2, 3-Dioxygenase in Human Dendritic Cells by a Cholera Toxin B Subunit—Proinsulin Vaccine
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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.

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: A novel fragment derived from the β chain of human fibrinogen, β43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo
Article Snippet: Paragraph title: Western blotting for phosphorylated Akt ... After treatment the cells were washed twice in PBS and suspended in a triple cell lysis buffer (50 nM Tris-HCI (pH 8.5), 150 nM NaCl, 0.1% SDS, 1% nonident-P40, 0.5% sodium deoxycholate and a complete protease inhibitor tablet (Roche, Mannheim, Germany)) and placed on ice for 20 min before centrifugation.

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.

Acid Assay:

Article Title: A novel fragment derived from the β chain of human fibrinogen, β43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo
Article Snippet: After treatment the cells were washed twice in PBS and suspended in a triple cell lysis buffer (50 nM Tris-HCI (pH 8.5), 150 nM NaCl, 0.1% SDS, 1% nonident-P40, 0.5% sodium deoxycholate and a complete protease inhibitor tablet (Roche, Mannheim, Germany)) and placed on ice for 20 min before centrifugation. .. Protein concentrations of the supernatants were determined using the bicinchoninic acid assay.

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: Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains *
Article Snippet: COS-1 transfections were carried out for 24 h in p60 dishes using GeneCellin (BioCellChallenge, Toulon, France) according to the protocol of the manufacturer. .. Cells were lysed with radio-immunoprecipitation assay buffer (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)).

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.

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 .

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: A novel fragment derived from the β chain of human fibrinogen, β43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo
Article Snippet: .. After treatment the cells were washed twice in PBS and suspended in a triple cell lysis buffer (50 nM Tris-HCI (pH 8.5), 150 nM NaCl, 0.1% SDS, 1% nonident-P40, 0.5% sodium deoxycholate and a complete protease inhibitor tablet (Roche, Mannheim, Germany)) and placed on ice for 20 min before centrifugation. .. Protein concentrations of the supernatants were determined using the bicinchoninic acid assay.

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: Sheddase Activity of Tumor Necrosis Factor-Alpha Converting Enzyme is Increased and Prognostically Valuable in Head and Neck Cancer
Article Snippet: .. Briefly, cultured cells (1 × 106 ) were lysed in 50 μl of lysis buffer, and fresh tissues (50 mg) were homogenized using the tissue grinder system (Fisher Scientific, Pittsburgh, PA) and lysed in 500 μl of lysis buffer on ice, for 1 h. The lysis buffer was composed of Tris-HCl (50 mM), NaCl (150 mM), SDS (0.1%), sodium deoxycholate (1%), Nonidet P40 (1%) and Triton X-100 (1%), and EDTA-free protease inhibitor cocktail (Roche Applied Science). .. TACE enzymatic assay was performed with the 150 μl mixture of 10 μg lysate proteins, assay buffer [Tris-HCl (50 mM, pH 7.4), NaCl (25 mM), glycerol (4%), EDTA-free protease inhibitor cocktail (Roche Applied Science)], and 50 μM of TACE-specific fluorophore/quencher-capped-end substrate with the amino-acid sequence Abz-Leu-Ala-Gln- Ala-Val -Arg-Ser-Ser-Ser-Arg-Dap(Dnp)-NH2 containing the TACE specific cleavage site Ala-Val (Peptide International, Louisville, KY).

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: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
Article Snippet: .. In brief, uninfected or infected HeLa cells were washed twice with ice-cold PBS 27 hpi and lysed in cell lysis buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl2 , 0.5% Nonidet P40, 5 mM dithiothreitol (DTT), protease inhibitor (cOmplete Tablets, Mini Easypack, Roche) and 40 U/ml RNase inhibitor (Recombinant RNasin Ribonuclease Inhibitor, Promega) for 30 min at 4°C on an overhead tumbler. .. Cell debris was removed by centrifugation at 20,000 g for 30 min at 4°C.

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: Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains *
Article Snippet: .. Cells were lysed with radio-immunoprecipitation assay buffer (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)). .. Immunoprecipitations were performed with antibodies against the TCR Cα domain (TCR1145) or Cβ domain (TCR1151) (Thermo Fisher Scientific, Rockford, IL), the FLAG tag (F7425, Sigma), BiP , or the HA tag (produced in our laboratory).

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).

Cell Culture:

Article Title: Sheddase Activity of Tumor Necrosis Factor-Alpha Converting Enzyme is Increased and Prognostically Valuable in Head and Neck Cancer
Article Snippet: .. Briefly, cultured cells (1 × 106 ) were lysed in 50 μl of lysis buffer, and fresh tissues (50 mg) were homogenized using the tissue grinder system (Fisher Scientific, Pittsburgh, PA) and lysed in 500 μl of lysis buffer on ice, for 1 h. The lysis buffer was composed of Tris-HCl (50 mM), NaCl (150 mM), SDS (0.1%), sodium deoxycholate (1%), Nonidet P40 (1%) and Triton X-100 (1%), and EDTA-free protease inhibitor cocktail (Roche Applied Science). .. TACE enzymatic assay was performed with the 150 μl mixture of 10 μg lysate proteins, assay buffer [Tris-HCl (50 mM, pH 7.4), NaCl (25 mM), glycerol (4%), EDTA-free protease inhibitor cocktail (Roche Applied Science)], and 50 μM of TACE-specific fluorophore/quencher-capped-end substrate with the amino-acid sequence Abz-Leu-Ala-Gln- Ala-Val -Arg-Ser-Ser-Ser-Arg-Dap(Dnp)-NH2 containing the TACE specific cleavage site Ala-Val (Peptide International, Louisville, KY).

Article Title: Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains *
Article Snippet: Paragraph title: Cell Culture Experiments ... Cells were lysed with radio-immunoprecipitation assay buffer (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)).

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).

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.

Sequencing:

Article Title: Sheddase Activity of Tumor Necrosis Factor-Alpha Converting Enzyme is Increased and Prognostically Valuable in Head and Neck Cancer
Article Snippet: Briefly, cultured cells (1 × 106 ) were lysed in 50 μl of lysis buffer, and fresh tissues (50 mg) were homogenized using the tissue grinder system (Fisher Scientific, Pittsburgh, PA) and lysed in 500 μl of lysis buffer on ice, for 1 h. The lysis buffer was composed of Tris-HCl (50 mM), NaCl (150 mM), SDS (0.1%), sodium deoxycholate (1%), Nonidet P40 (1%) and Triton X-100 (1%), and EDTA-free protease inhibitor cocktail (Roche Applied Science). .. TACE enzymatic assay was performed with the 150 μl mixture of 10 μg lysate proteins, assay buffer [Tris-HCl (50 mM, pH 7.4), NaCl (25 mM), glycerol (4%), EDTA-free protease inhibitor cocktail (Roche Applied Science)], and 50 μM of TACE-specific fluorophore/quencher-capped-end substrate with the amino-acid sequence Abz-Leu-Ala-Gln- Ala-Val -Arg-Ser-Ser-Ser-Arg-Dap(Dnp)-NH2 containing the TACE specific cleavage site Ala-Val (Peptide International, Louisville, KY).

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.

Recombinant:

Article Title: Sheddase Activity of Tumor Necrosis Factor-Alpha Converting Enzyme is Increased and Prognostically Valuable in Head and Neck Cancer
Article Snippet: Briefly, cultured cells (1 × 106 ) were lysed in 50 μl of lysis buffer, and fresh tissues (50 mg) were homogenized using the tissue grinder system (Fisher Scientific, Pittsburgh, PA) and lysed in 500 μl of lysis buffer on ice, for 1 h. The lysis buffer was composed of Tris-HCl (50 mM), NaCl (150 mM), SDS (0.1%), sodium deoxycholate (1%), Nonidet P40 (1%) and Triton X-100 (1%), and EDTA-free protease inhibitor cocktail (Roche Applied Science). .. Recombinant TACE (rTACE) (1-100 ng), a specific inhibitor of TACE (recombinant tissue inhibitor of metalloproteases-3 - rTIMP-3,100 mM), a specific inhibitor of matrix metalloproteases (MMPs) (rTIMP-1, 100 mM) (R & D Systems), and a general inhibitor of metalloproteases (EDTA, 10 mM) were used to assess the specificity of the TACE sheddase assay.

Article Title: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
Article Snippet: .. In brief, uninfected or infected HeLa cells were washed twice with ice-cold PBS 27 hpi and lysed in cell lysis buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl2 , 0.5% Nonidet P40, 5 mM dithiothreitol (DTT), protease inhibitor (cOmplete Tablets, Mini Easypack, Roche) and 40 U/ml RNase inhibitor (Recombinant RNasin Ribonuclease Inhibitor, Promega) for 30 min at 4°C on an overhead tumbler. .. Cell debris was removed by centrifugation at 20,000 g for 30 min at 4°C.

Nucleic Acid Electrophoresis:

Article Title: A novel fragment derived from the β chain of human fibrinogen, β43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo
Article Snippet: After treatment the cells were washed twice in PBS and suspended in a triple cell lysis buffer (50 nM Tris-HCI (pH 8.5), 150 nM NaCl, 0.1% SDS, 1% nonident-P40, 0.5% sodium deoxycholate and a complete protease inhibitor tablet (Roche, Mannheim, Germany)) and placed on ice for 20 min before centrifugation. .. Equal amounts of protein extracts (20 μ g) were separated by SDS–polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose membranes and exposed to anti-Akt or phospho-Akt (BD Biosciences).

Fluorescence:

Article Title: Sheddase Activity of Tumor Necrosis Factor-Alpha Converting Enzyme is Increased and Prognostically Valuable in Head and Neck Cancer
Article Snippet: Briefly, cultured cells (1 × 106 ) were lysed in 50 μl of lysis buffer, and fresh tissues (50 mg) were homogenized using the tissue grinder system (Fisher Scientific, Pittsburgh, PA) and lysed in 500 μl of lysis buffer on ice, for 1 h. The lysis buffer was composed of Tris-HCl (50 mM), NaCl (150 mM), SDS (0.1%), sodium deoxycholate (1%), Nonidet P40 (1%) and Triton X-100 (1%), and EDTA-free protease inhibitor cocktail (Roche Applied Science). .. The enzymatic reaction was induced by the incubation of the lysate/substrate mixture at room temperature for 3 h. The fluorescence was measured in a LS55 Luminescence Spectrometer (Perkin-Elmer, Fremont, CA) using an excitation wavelength (λex ) of 320 nm and an emission wavelength (λem ) of 420 nm.

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). ..

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).

Labeling:

Article Title: Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains *
Article Snippet: Labeling was carried out in the presence of 10 m m DTT for DTT washout experiments. .. Cells were lysed with radio-immunoprecipitation assay buffer (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)).

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.

Immunostaining:

Article Title: Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency
Article Snippet: Paragraph title: Immunostaining ... Cells were permeabilized in 0.2% Nonidet P40 (Roche) and blocked in 10% goat serum.

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: Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains *
Article Snippet: Prior to lysis, cells were washed twice in ice-cold PBS supplemented with 20 m m N -ethylmaleimide when samples were to be run on non-reducing SDS-PAGE gels. .. Cells were lysed with radio-immunoprecipitation assay buffer (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)).

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 .

Plasmid Preparation:

Article Title: Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency
Article Snippet: Cells were permeabilized in 0.2% Nonidet P40 (Roche) and blocked in 10% goat serum. .. Cells were permeabilized in 0.2% Nonidet P40 (Roche) and blocked in 10% goat serum.

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.

Article Title: A novel fragment derived from the β chain of human fibrinogen, β43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo
Article Snippet: After treatment the cells were washed twice in PBS and suspended in a triple cell lysis buffer (50 nM Tris-HCI (pH 8.5), 150 nM NaCl, 0.1% SDS, 1% nonident-P40, 0.5% sodium deoxycholate and a complete protease inhibitor tablet (Roche, Mannheim, Germany)) and placed on ice for 20 min before centrifugation. .. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham, Little Chalfont, Buckinghamshire, UK), and band intensity quantified using densitometry software (Bio-Rad, Hemel Hempstead, Hertfordshire, UK).

Co-Immunoprecipitation Assay:

Article Title: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
Article Snippet: Paragraph title: Co-immunoprecipitation (Co-IP) of human argonaute protein complexes ... In brief, uninfected or infected HeLa cells were washed twice with ice-cold PBS 27 hpi and lysed in cell lysis buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl2 , 0.5% Nonidet P40, 5 mM dithiothreitol (DTT), protease inhibitor (cOmplete Tablets, Mini Easypack, Roche) and 40 U/ml RNase inhibitor (Recombinant RNasin Ribonuclease Inhibitor, Promega) for 30 min at 4°C on an overhead tumbler.

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.

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: Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains *
Article Snippet: .. Cells were lysed with radio-immunoprecipitation assay buffer (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)). .. Immunoprecipitations were performed with antibodies against the TCR Cα domain (TCR1145) or Cβ domain (TCR1151) (Thermo Fisher Scientific, Rockford, IL), the FLAG tag (F7425, Sigma), BiP , or the HA tag (produced in our laboratory).

Incubation:

Article Title: A novel fragment derived from the β chain of human fibrinogen, β43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo
Article Snippet: The cells were then incubated with peptides for 3 h before a 5-min incubation with VEGF (10 ng ml−1 ). .. After treatment the cells were washed twice in PBS and suspended in a triple cell lysis buffer (50 nM Tris-HCI (pH 8.5), 150 nM NaCl, 0.1% SDS, 1% nonident-P40, 0.5% sodium deoxycholate and a complete protease inhibitor tablet (Roche, Mannheim, Germany)) and placed on ice for 20 min before 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. .. Samples were incubated on ice for 10 min, centrifuged as above, salt extraction repeated once, then pellets washed twice in PEME.

Article Title: Sheddase Activity of Tumor Necrosis Factor-Alpha Converting Enzyme is Increased and Prognostically Valuable in Head and Neck Cancer
Article Snippet: Briefly, cultured cells (1 × 106 ) were lysed in 50 μl of lysis buffer, and fresh tissues (50 mg) were homogenized using the tissue grinder system (Fisher Scientific, Pittsburgh, PA) and lysed in 500 μl of lysis buffer on ice, for 1 h. The lysis buffer was composed of Tris-HCl (50 mM), NaCl (150 mM), SDS (0.1%), sodium deoxycholate (1%), Nonidet P40 (1%) and Triton X-100 (1%), and EDTA-free protease inhibitor cocktail (Roche Applied Science). .. The enzymatic reaction was induced by the incubation of the lysate/substrate mixture at room temperature for 3 h. The fluorescence was measured in a LS55 Luminescence Spectrometer (Perkin-Elmer, Fremont, CA) using an excitation wavelength (λex ) of 320 nm and an emission wavelength (λem ) of 420 nm.

Article Title: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
Article Snippet: In brief, uninfected or infected HeLa cells were washed twice with ice-cold PBS 27 hpi and lysed in cell lysis buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl2 , 0.5% Nonidet P40, 5 mM dithiothreitol (DTT), protease inhibitor (cOmplete Tablets, Mini Easypack, Roche) and 40 U/ml RNase inhibitor (Recombinant RNasin Ribonuclease Inhibitor, Promega) for 30 min at 4°C on an overhead tumbler. .. 15 μg of anti-pan Ago mAb clone 2A8 (Merck Millipore) or anti-Rep mAb 76–3 (Progen) were added to the pre-washed beads, respectively, and incubated for 6 hours at 4°C under constant rotation.

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. .. Proteins were transferred to nitrocellulose membranes, blocked for 1 h at room temperature (RT) with 5 % nonfat dried milk in tween-phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 5 mM Na2 HPO4 -7H2 0, 1.4 mM KH2 PO4 , and 0.1 % Tween; pH 7.4) (PBS-T), and incubated overnight at 4 °C with anti-c-myc antibody (Invitrogen) at 1:2500 or anti-α-tubulin antibody (Sigma-Aldrich) at 1:10,000.

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: Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency
Article Snippet: Cells were permeabilized in 0.2% Nonidet P40 (Roche) and blocked in 10% goat serum. .. Cells were incubated in the following primary antibodies at 4°C overnight: Oct3/4 (Santa Cruz Biotechnology), Sox2 (R & D Systems), Nanog (Abcam), SSEA1 and SSEA4 (Developmental Studies Hybridoma Bank), TRA-1-60 and TRA-1-81 (Millipore, Inc.), and GFP (Clontech).

Produced:

Article Title: Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains *
Article Snippet: Cells were lysed with radio-immunoprecipitation assay buffer (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)). .. Immunoprecipitations were performed with antibodies against the TCR Cα domain (TCR1145) or Cβ domain (TCR1151) (Thermo Fisher Scientific, Rockford, IL), the FLAG tag (F7425, Sigma), BiP , or the HA tag (produced in our laboratory).

Immunoprecipitation:

Article Title: Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains *
Article Snippet: Cells were lysed with radio-immunoprecipitation assay buffer (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)). .. Immunoprecipitated proteins were washed three times with radio-immunoprecipitation assay buffer or Nonidet P-40 washing buffer (for chaperone co-immunoprecipitations 50 m m Tris/HCl (pH 7.5), 400 m m NaCl, 0.5% Nonidet P40 substitute, and 0.5% sodium deoxycholate) and eluted with Laemmli buffer for 5 min at 95 °C.

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.

FLAG-tag:

Article Title: Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains *
Article Snippet: Cells were lysed with radio-immunoprecipitation assay buffer (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)). .. Immunoprecipitations were performed with antibodies against the TCR Cα domain (TCR1145) or Cβ domain (TCR1151) (Thermo Fisher Scientific, Rockford, IL), the FLAG tag (F7425, Sigma), BiP , or the HA tag (produced in our laboratory).

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: A novel fragment derived from the β chain of human fibrinogen, β43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo
Article Snippet: .. After treatment the cells were washed twice in PBS and suspended in a triple cell lysis buffer (50 nM Tris-HCI (pH 8.5), 150 nM NaCl, 0.1% SDS, 1% nonident-P40, 0.5% sodium deoxycholate and a complete protease inhibitor tablet (Roche, Mannheim, Germany)) and placed on ice for 20 min before centrifugation. .. Protein concentrations of the supernatants were determined using the bicinchoninic acid assay.

Article Title: Sheddase Activity of Tumor Necrosis Factor-Alpha Converting Enzyme is Increased and Prognostically Valuable in Head and Neck Cancer
Article Snippet: .. Briefly, cultured cells (1 × 106 ) were lysed in 50 μl of lysis buffer, and fresh tissues (50 mg) were homogenized using the tissue grinder system (Fisher Scientific, Pittsburgh, PA) and lysed in 500 μl of lysis buffer on ice, for 1 h. The lysis buffer was composed of Tris-HCl (50 mM), NaCl (150 mM), SDS (0.1%), sodium deoxycholate (1%), Nonidet P40 (1%) and Triton X-100 (1%), and EDTA-free protease inhibitor cocktail (Roche Applied Science). .. TACE enzymatic assay was performed with the 150 μl mixture of 10 μg lysate proteins, assay buffer [Tris-HCl (50 mM, pH 7.4), NaCl (25 mM), glycerol (4%), EDTA-free protease inhibitor cocktail (Roche Applied Science)], and 50 μM of TACE-specific fluorophore/quencher-capped-end substrate with the amino-acid sequence Abz-Leu-Ala-Gln- Ala-Val -Arg-Ser-Ser-Ser-Arg-Dap(Dnp)-NH2 containing the TACE specific cleavage site Ala-Val (Peptide International, Louisville, KY).

Article Title: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
Article Snippet: .. In brief, uninfected or infected HeLa cells were washed twice with ice-cold PBS 27 hpi and lysed in cell lysis buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl2 , 0.5% Nonidet P40, 5 mM dithiothreitol (DTT), protease inhibitor (cOmplete Tablets, Mini Easypack, Roche) and 40 U/ml RNase inhibitor (Recombinant RNasin Ribonuclease Inhibitor, Promega) for 30 min at 4°C on an overhead tumbler. .. Cell debris was removed by centrifugation at 20,000 g for 30 min at 4°C.

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: Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains *
Article Snippet: .. Cells were lysed with radio-immunoprecipitation assay buffer (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 1.0% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA) or Nonidet P-40 lysis buffer in the case of chaperone co-immunoprecipitation experiments (50 m m Tris/HCl (pH 7.5), 150 m m NaCl, 0.5% Nonidet P40 substitute, 0.5% sodium deoxycholate, 0.1 m m PMSF, and 1× Roche complete protease inhibitor without EDTA, supplemented with 10 units/ml Apyrase for BiP interaction studies (Sigma-Aldrich, St. Louis, MO)). .. Immunoprecipitations were performed with antibodies against the TCR Cα domain (TCR1145) or Cβ domain (TCR1151) (Thermo Fisher Scientific, Rockford, IL), the FLAG tag (F7425, Sigma), BiP , or the HA tag (produced in our laboratory).

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: Autophagy links antimicrobial activity with antigen presentation in Langerhans cells
Article Snippet: .. LCDC, 1 × 106 per condition, were collected in NP-40 lysis buffer (50 mM Tris [pH 7.4], 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3 VO4 , 1% Nonidet P40 [NP40],0.02% NaN3 and 1 mM PMSF) containing complete protease inhibitors (Roche Applied Science). .. Total protein from cell lysates were quantified using Bradford Assay.

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).

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.

Serum Depletion:

Article Title: A novel fragment derived from the β chain of human fibrinogen, β43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo
Article Snippet: Western blotting for phosphorylated Akt Human dermal microvascular endothelial cells were plated down into six-well plates and grown to near confluence before serum depletion (EGM+1%FCS) overnight. .. After treatment the cells were washed twice in PBS and suspended in a triple cell lysis buffer (50 nM Tris-HCI (pH 8.5), 150 nM NaCl, 0.1% SDS, 1% nonident-P40, 0.5% sodium deoxycholate and a complete protease inhibitor tablet (Roche, Mannheim, Germany)) and placed on ice for 20 min before centrifugation.

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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
    Price from $9.99 to $1999.99
    ripa buffer - by Bioz Stars, 2020-02
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    99
    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
    https://www.bioz.com/result/nonidet p40/product/Roche
    Average 99 stars, based on 109 article reviews
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    80
    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: 80/100, 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