Journal: The Journal of Biological Chemistry

Article Title: The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein *

doi: 10.1074/jbc.M115.710772

Figure Lengend Snippet: Multidimensional electrophoretic resolution of NOX4-HEK293 membranes by two-dimensional BN/SDS-PAGE. A , 15,000 × g membrane pellet from differential centrifugation of NOX4-HEK293 (N4) cells was solubilized with digitonin (6 g/g of protein) and separated by one-dimensional BN-PAGE (3 > 16% acrylamide) with subsequent two-dimensional separation by Tricine-SDS-PAGE with 10% acrylamide. Representative BN-PAGE gel and silver staining of the two-dimensional BN/SDS-PAGE gel are shown. The two-dimensional BN/SDS-PAGE gel was blotted and sequential antibody detection was performed. Protein complexes of the mitochondria (bovine heart mitochondria; BHM ) were used as size standard. B , representative two-dimensional BN/SDS-PAGE blots of NOX4 solubilized with different detergents (digitonin, DDM ( n -dodecyl β- d -maltopyranoside), or Triton X-100) and concentrations as indicated.

Article Snippet: Cells were fixed in 4% phosphate-buffered formaldehyde solution, permeabilized with 0.05% Triton X-100, blocked, and incubated overnight with rabbit monoclonal antibodies against NOX4 (provided by one of the co-authors (P.J.-D.)) and CANX antibody (Santa Cruz, number sc-6465) as well as negative and positive controls to validate the assay (data not shown).

Techniques: SDS Page, Centrifugation, Polyacrylamide Gel Electrophoresis, Silver Staining

Journal: The Journal of Biological Chemistry

Article Title: The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein *

doi: 10.1074/jbc.M115.710772

Figure Lengend Snippet: Determination of interaction partners using qCo-IP and standard Co-IP with subsequent Western blot analysis. HEK293 ( Ctl ) and NOX4-HEK293 ( N4 ) cells were cultured in normal or heavy labeled (*) SILAC medium ( 13 C-R; 13 C 15 N-K), lysed with either 1% digitonin ( n = 2) in TBS or 100,000 × g total membrane pellets ( n = 3) were solubilized with digitonin (6 g/g of protein). Samples were used for NOX4 qCo-IP with magnetic beads and subsequent MS detection. A , logarithmic normalized L/H ratios of forward (N4/Ctl*) and H/L in reverse (N4*/Ctl) experiment analyzed by MS were exemplary plotted against each other (experiment B2 ). Background proteins show a log 2 (H/L) ratio around 0 in both experiments, specific interactors have a high log 2 (L/H) in the forward and a high log 2 (H/L) ratio in the reverse experiment. NOX4 is highlighted in red and proteins with a threshold over 0.5 in blue . Proteins of interest are labeled B , Venn diagram compares proteins found in different experiments with a threshold > 0.5 ( n = 2; n = 3). C , table showing proteins with threshold of > 0.5 found in more than one experiment per preparation. D and E , representative Western blot for NOX4 in initial ( Ini ), preclearing ( Pre ), immunoprecipitation ( IP ), and post-IP fraction after lysis with 1% Triton -X-100 or digitonin of HEK293 ( Ctl ) and NOX4-HEK293 ( N4 ). Co-IP was performed with anti-HSPA5 ( D ) or anti-CANX ( E ) antibody and Sepharose beads, n ≥ 3. F , representative Western blot of CANX in Ini, Pre, IP, and post-IP fraction after Co-IP with NOX4 antibody. Ctl and N4 cells (used for SILAC-based Co-IP), lysed with either 1% digitonin in TBS ( n = 2) or 100,000 × g membrane pellets were solubilized with digitonin (6 g/g of protein; n = 3). IB , immunoblot.

Article Snippet: Cells were fixed in 4% phosphate-buffered formaldehyde solution, permeabilized with 0.05% Triton X-100, blocked, and incubated overnight with rabbit monoclonal antibodies against NOX4 (provided by one of the co-authors (P.J.-D.)) and CANX antibody (Santa Cruz, number sc-6465) as well as negative and positive controls to validate the assay (data not shown).

Techniques: Co-Immunoprecipitation Assay, Western Blot, CTL Assay, Cell Culture, Labeling, Magnetic Beads, Mass Spectrometry, Immunoprecipitation, Lysis

Journal: Infection and Immunity

Article Title: Cholesterol Depletion Reduces Entry of Campylobacter jejuni Cytolethal Distending Toxin and Attenuates Intoxication of Host Cells ▿

doi: 10.1128/IAI.05175-11

Figure Lengend Snippet: Cholesterol depletion in CHO-K1 cells by treatment with MβCD. (A) CHO-K1 cells were treated with 10 mM MβCD at 37°C and incubated for the indicated times. The cells were harvested and subjected to cold detergent extraction using 1% Triton X-100, followed by centrifugation to isolate the DRM fraction. The prepared total cell lysates and DRM fraction then were analyzed for cholesterol concentration as described in Materials and Methods. (B) CHO-K1 cells were treated with various concentrations of MβCD (0, 2.5, 5, 10, and 20 mM) for 1 h. Whole-cell lysates and the DRM fraction then were prepared for cholesterol level analysis. (C) Cell viability was barely influenced after treatment with 0 to 20 mM MβCD, as determined by the trypan blue exclusion assay. The data represent the means and standard deviations from three independent experiments. An asterisk indicates P

Article Snippet: The cultured cells then were washed three times with PBS and fixed with 3.7% paraformaldehyde (Sigma-Aldrich) for 1 h. The cells were permeabilized with 0.1% Triton X-100 for 30 min and stained with anti-caveolin-1 antibody (BD Pharmingen), followed by being stained with Alexa Fluor 647-conjugated anti-rabbit IgG (Molecular Probes).

Techniques: Incubation, Centrifugation, Concentration Assay, Trypan Blue Exclusion Assay

Journal: Infection and Immunity

Article Title: Cholesterol Depletion Reduces Entry of Campylobacter jejuni Cytolethal Distending Toxin and Attenuates Intoxication of Host Cells ▿

doi: 10.1128/IAI.05175-11

Figure Lengend Snippet: CdtA and CdtC are enriched in detergent-resistant membrane (DRM) fractions. CHO-K1 cells were left untreated or treated with 10 mM MβCD for 1 h prior to incubation with C. jejuni CDT holotoxin (200 nM) for 2 h at 37°C. The cells then were subjected to cold detergent extraction using 1% Triton X-100, followed by centrifugation to separate the DRM and detergent-soluble (S) fractions. (A) Each fraction was subjected to Western blot analysis using antibodies against caveolin-1 and CD71 and individual antisera specific to CdtA, CdtB, and CdtC. The results are representative of one of three independent experiments. (B) The protein expression levels were analyzed using scanning densitometry. The protein expression levels represent the relative distribution (percent) of each protein within the DRM and soluble fractions. MβCD, methyl-β-cyclodextrin.

Article Snippet: The cultured cells then were washed three times with PBS and fixed with 3.7% paraformaldehyde (Sigma-Aldrich) for 1 h. The cells were permeabilized with 0.1% Triton X-100 for 30 min and stained with anti-caveolin-1 antibody (BD Pharmingen), followed by being stained with Alexa Fluor 647-conjugated anti-rabbit IgG (Molecular Probes).

Techniques: Incubation, Centrifugation, Western Blot, Expressing

Journal: Journal of neurochemistry

Article Title: LIPID CONTENT OF BRAIN, OF BRAIN MEMBRANE LIPID DOMAINS, AND OF NEURONS FROM ACID SPHINGOMYELINASE DEFICIENT MICE (ASMKO)

doi: 10.1111/j.1471-4159.2008.05591.x

Figure Lengend Snippet: Lipid and protein distribution in sucrose gradient fractions obtained from ASMKO mouse brain homogenate containing 3 mg (panel A), 1.3 mg (panel B) and 1mg of proteins (panel C) per 1 ml of Triton X-100 in lysis buffer, by ultracentrifugation

Article Snippet: Different final protein/detergent ratios were obtained adding Triton X-100 in TNEV to 1 to 6 mg of protein homogenates, to reach a final concentration of 1% Triton X-100 in 1 mL.

Techniques: Lysis

Journal: Journal of neurochemistry

Article Title: LIPID CONTENT OF BRAIN, OF BRAIN MEMBRANE LIPID DOMAINS, AND OF NEURONS FROM ACID SPHINGOMYELINASE DEFICIENT MICE (ASMKO)

doi: 10.1111/j.1471-4159.2008.05591.x

Figure Lengend Snippet: Lipid and protein distribution in sucrose gradient fractions obtained from WT mouse brain homogenate containing 3mg (panel A), 1.3 mg (panel B) and 1mg of proteins (panel C) per 1 ml of Triton X-100 in lysis buffer, by ultracentrifugation

Article Snippet: Different final protein/detergent ratios were obtained adding Triton X-100 in TNEV to 1 to 6 mg of protein homogenates, to reach a final concentration of 1% Triton X-100 in 1 mL.

Techniques: Lysis

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of TOC as a function of the reaction time under various conditions in the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 10 −3 mol·dm −3 Na 2 S 2 O 8 : air ( • ), air/UV (○), air/UV/TiO 2 (1 g dm −3 ) (•).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques:

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the Triton X-100 concentration as a function of the reaction time under various conditions in the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and ozonated at 3.5 × 10 −4 mol·dm −3 ·min −1 rate: air ( • ), air/UV (○) and air/UV/TiO 2 (1 g·dm −3 ) (•).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques: Concentration Assay

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the TOC as a function of the reaction time under various conditions in the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and ozonated at 3.5 × 10 −4 mol·dm −3 ·min −1 rate: air ( • ), air/UV (○) and air/UV/TiO 2 (1 g dm −3 ) (•).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques:

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: Intensity vs. irradiation time plots for the most abundant fragment ion of the starting components of Triton X-100 with molecular weights of 470 ( m / z = 399), 426 ( m / z = 355) ( A ) and 382 ( m / z = 311), 338 ( m / z = 267) and 294 ( m / z = 223) ( B ) in the photocatalysis of the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 TiO 2 .

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques: Irradiation

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the TOC as a function of the irradiation time at various pH values in the aerated system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 TiO 2 .

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques: Irradiation

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the Triton X-100 concentration as a function of the reaction time under various conditions in the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 TiO 2 : air ( • ), air/UV (○) and air/UV/TiO 2 (•).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques: Concentration Assay

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: Intensity vs. irradiation time plots for the most abundant fragment ion of characteristic intermediates with molecular weights of 206 ( m / z = 135), 148 ( m / z = 73) ( A ) and 118 ( m / z = 87) ( B ) in the photocatalysis of the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 TiO 2 .

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques: Irradiation

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the absorbance at 223 nm ( A ) and 275 nm ( B ) as a function of the reaction time under various conditions in the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 TiO 2 (ℓ = 1 cm): air ( • ), air/UV (○) and air/UV/TiO 2 (•).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques:

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the peak intensity as a function of the reaction time from the UHPLC chromatograms obtained during the photocatalysis of the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 TiO 2 : at a retention time of 11.41 min ( A ), 13.02 min ( B ) and 14.55 min ( C ).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques:

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the absorption spectrum (after removal of the suspended TiO 2 ) during the photocatalysis in the aerated system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 catalyst (ℓ = 1 cm).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques:

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the Triton X-100 concentration as a function of the reaction time under various conditions in the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 × 10 −3 mol·dm −3 Na 2 S 2 O 8 : air ( • ), air/UV (○) and air/UV/TiO 2 (1 g dm −3 ) (•).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques: Concentration Assay

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the TOC as a function of time under various conditions in the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 TiO 2 : air ( • ), air/UV (○) and air/UV/TiO 2 (•).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques:

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The peak intensity as a function of the retention time, taken from the UHPLC chromatograms during the photocatalysis of the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 TiO 2 , after 0, 10, 20 and 40 min of reaction time.

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques:

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the TOC as a function of time in the system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 TiO 2 : for the whole system (•), for the unreacted surfactant (○) and for the intermediates ( • ).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques:

Journal: Materials

Article Title: TiO2-Mediated Photocatalytic Mineralization of a Non-Ionic Detergent: Comparison and Combination with Other Advanced Oxidation Procedures

doi: 10.3390/ma8010231

Figure Lengend Snippet: The change of the emission intensity (after removal of the suspended TiO 2 ) during the photocatalysis in the aerated system containing 2 × 10 −4 mol·dm −3 Triton X-100 and 1 g·dm −3 catalyst (ℓ = 1 cm, λ ex =277 nm, λ em =302 nm).

Article Snippet: Degradation of Triton X-100 was followed by 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a binary gradient pump, automatic injector, column thermostat, DAD detector and Chemstation data acquisition system.

Techniques:

Journal: PLoS ONE

Article Title: Preclinical Evaluation of 4-Methylthiobutyl Isothiocyanate on Liver Cancer and Cancer Stem Cells with Different p53 Status

doi: 10.1371/journal.pone.0070846

Figure Lengend Snippet: Cell cycle dependency of MTBITC-induced cytotoxicity and apoptosis induction in HCC cells. (a–d) influence of cell number on sensitivity to MTBITC-induced growth arrest and cytotoxicity, as assessed by DNA content analysis (a, b), neutral red retention (c) or subG1 peak analysis (d) of HCC cells. 0.1% DMSO was used as solvent control, 0.01% triton X-100 as positive control (+). (e and f) impact of G0/1 phase synchronization on cell sensitivity to MTBITC in HepG2 cells, as determined by DNA content analysis. (e) cells were treated with 2% DMSO for 96 h and subsequently treated with 25 µM MTBITC or 0.1% DMSO for another 24 h. (f) cells were starved for 96 h and subsequently treated with 25 µM MTBITC or 0.1% DMSO for another 24 h, while adding varying amounts of serum. Bars are mean+SD, (n = 3).

Article Snippet: CaCl2 , Glucose, EGTA, Acetic acid (purity 100%), Roti®-Phenol/Chloroform/Isoamylalkohol and Chloroform/Isoamylalkohol were acquired from Carl Roth (Karlsruhe, Germany), Camptothecin (CPT) from Tocris (Eching, Germany), Caspase 3/7 GLO reagent from Promega (Mannheim, Germany) and Triton X-100 from Merck (Mannheim, Germany).

Techniques: Positive Control

Journal: PLoS ONE

Article Title: Preclinical Evaluation of 4-Methylthiobutyl Isothiocyanate on Liver Cancer and Cancer Stem Cells with Different p53 Status

doi: 10.1371/journal.pone.0070846

Figure Lengend Snippet: MTBITC selectively triggers apoptosis in liver tumor cells. Impact of MTBITC on apoptosis (a, c, and e) or necrosis (b, d and f) induction in malignant and healthy cells. Distinct apoptosis induction was assessed using caspase 3/7 activity after cell treatment with MTBITC for 12 to 72 h. Positive control (+): 10 µM CPT or staurosporine (a, c), 1 µg/ml actinomycin D/100 ng/ml TNF (ActD/TNF) (e). Necrosis induction was quantified using specific uptake of PI in isolated cells (b and d) or LDH release from PCLS (f). Positive control (+): 0.2% triton X-100. Results were expressed relative to the solvent (0.1% DMSO). Bars are mean+SD, (number of independent experiments is given below the figures; experiments conducted on PCLS were conducted in triplicate.).

Article Snippet: CaCl2 , Glucose, EGTA, Acetic acid (purity 100%), Roti®-Phenol/Chloroform/Isoamylalkohol and Chloroform/Isoamylalkohol were acquired from Carl Roth (Karlsruhe, Germany), Camptothecin (CPT) from Tocris (Eching, Germany), Caspase 3/7 GLO reagent from Promega (Mannheim, Germany) and Triton X-100 from Merck (Mannheim, Germany).

Techniques: Activity Assay, Positive Control, Cycling Probe Technology, Isolation

Journal: PLoS ONE

Article Title: Preclinical Evaluation of 4-Methylthiobutyl Isothiocyanate on Liver Cancer and Cancer Stem Cells with Different p53 Status

doi: 10.1371/journal.pone.0070846

Figure Lengend Snippet: MTBITC selectively kills liver tumor cells. MTBITC treatment selectively attenuates cell survival of liver tumor cells (a) as compared to primary hepatocytes (b). Cell viability was assessed using the NR retention assay after exposure to MTBITC for 24–72 h. Results were expressed relative to control (0.1% DMSO), bars are mean+SD, (number of independent experiments is given below the figures). Positive control (+), 0.01% Triton X-100. ** ≤p

Article Snippet: CaCl2 , Glucose, EGTA, Acetic acid (purity 100%), Roti®-Phenol/Chloroform/Isoamylalkohol and Chloroform/Isoamylalkohol were acquired from Carl Roth (Karlsruhe, Germany), Camptothecin (CPT) from Tocris (Eching, Germany), Caspase 3/7 GLO reagent from Promega (Mannheim, Germany) and Triton X-100 from Merck (Mannheim, Germany).

Techniques: Positive Control

Journal: PLoS ONE

Article Title: Streptococcus sanguinis induces neutrophil cell death by production of hydrogen peroxide

doi: 10.1371/journal.pone.0172223

Figure Lengend Snippet: H 2 O 2 produced by S . sanguinis showed cytotoxicity against neutrophils. Human neutrophils were mixed with the S . sanguinis WT, KO, or Wr strain at an MOI of 10. As a positive control, PMA was added at concentrations of 200 nM. Following incubation for 1 or 3 h, LDH released into culture supernatant was examined. Cytotoxicity was determined by relative absorbance, with the absorbance of cells lysed with PBS containing 0.25% Triton X-100 set at 100%. Data are presented as the mean ± SD from 3 independent experiments. Statistically significant differences were evaluated using one-way ANOVA and Tukey’s multiple comparison test. * p

Article Snippet: The absorbance of cell lysates treated with PBS containing 0.25% Triton X-100 (Wako) was set at 100%.

Techniques: Produced, Positive Control, Incubation

Journal: Scientific Reports

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography

doi: 10.1038/s41598-018-22359-w

Figure Lengend Snippet: Association of NPM1 with RNA in the high molecular weight fractions. ( A ) Triton X-100 soluble and insoluble cell lysates were subjected to SEC. The indicated fractions were pooled, then immunoprecipitated with anti-NPM. NPM1 and RNA were detected by immunoblot and ethidium bromide staining, respectively. ( B ) HeLa cell lysates prepared as in Fig.  2A were treated with or without RNase A, then subjected to SEC. Fractions containing the four NPM1 groups identified in Fig.  3A are underlined.

Article Snippet: Immunoprecipitates were eluted with PBS containing 1% Triton X-100 and 2 M guanidine-HCl (Nacalai Tesque).

Techniques: Molecular Weight, Size-exclusion Chromatography, Immunoprecipitation, Staining

Journal: Scientific Reports

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography

doi: 10.1038/s41598-018-22359-w

Figure Lengend Snippet: Distribution of NPM1 by simple cell fractionation. ( A ) Summary of the process of cell fractionation. ( B ) Distribution of NPM1 in the sample prepared in A. ( C ) Immunofluorescence of endogenous NPM1. ( Upper panel ) HeLa cells were fixed with formaldehyde, then permeabilized using Triton X-100. ( Lower panel ) HeLa cells were permeabilized using Triton X-100, then fixed with formaldehyde. Scale bar, 10 μm.

Article Snippet: Immunoprecipitates were eluted with PBS containing 1% Triton X-100 and 2 M guanidine-HCl (Nacalai Tesque).

Techniques: Cell Fractionation, Immunofluorescence

Journal: Scientific Reports

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography

doi: 10.1038/s41598-018-22359-w

Figure Lengend Snippet: The main component of cellular NPM1 complex is NPM1 and its variant. Both the Triton X-100 soluble and insoluble lysates from HeLa cells were subjected to SEC. Fractions 15 and 16 from the soluble sample (for Group 1), fractions 19 to 21 from the soluble sample (for Group 2), and fractions 15 and 16 from the insoluble sample (for Group 3) were separately pooled. After treatment with or without RNase A, each pooled sample was immunoprecipitated with anti-NPM antibody. Proteins were detected by CBB staining ( Upper panel ) and immunoblot using anti-NPM antibody ( Lower panel ).

Article Snippet: Immunoprecipitates were eluted with PBS containing 1% Triton X-100 and 2 M guanidine-HCl (Nacalai Tesque).

Techniques: Variant Assay, Size-exclusion Chromatography, Immunoprecipitation, Staining

Journal: Scientific Reports

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography

doi: 10.1038/s41598-018-22359-w

Figure Lengend Snippet: The association of NPM1 with RNA is attenuated in a NPM1c-positive cell line. ( A ) RNA was extracted from HL60 (expressing NPM1 (WT) ) and OCI-AML3 (expressing NPM1 (WT) and NPM1c ) cells. The expression of NPM1 (WT) and NPM1c was detected by RT-PCR. ( B ) Distribution of NPM1 in HL60 and OCI-AML3 cells prepared as in Fig.  2A . Then, NPM1 (WT) and NPM1c were detected by Western blotting. ( C ) HL60 and OCI-AML3 cells were lysed with buffer containing Triton X-100, SDS, and DOC. The lysate was immunoprecipitated with antibody against NPM1. NPM1 (WT) and NPM1c were detected by Western blotting with the indicated antibodies. ( D,E ) Triton X-100 soluble and insoluble lysates prepared from HL60 ( D ) and OCI-AML3 ( E ) cells were subjected to SEC. NPM1 (WT) and NPM1c were detected by Western blotting. RNA was extracted from each fraction and detected by ethidium bromide staining. ( F) Interaction of NPM1 complex with RNA. The Triton X-100 soluble lysates from HL60 and OCI-AML cells were immunoprecipitated with anti-NPM antibody. NPM1 and extracted RNA were detected by immunoblot and ethidium bromide staining, respectively. ( G ) Quantification of the data from panel F. The normalized RNA levels coprecipitated with NPM1 are shown. The data represent the average of three independent experiments ± SEM for each condition.

Article Snippet: Immunoprecipitates were eluted with PBS containing 1% Triton X-100 and 2 M guanidine-HCl (Nacalai Tesque).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Size-exclusion Chromatography, Staining

Journal: Scientific Reports

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography

doi: 10.1038/s41598-018-22359-w

Figure Lengend Snippet: Elution profiles of endogenous NPM1 in HeLa cells. ( A ) ( Upper panel ) Elution profiles of Triton X-100 soluble NPM1 and RNA. ( Lower panel ) Elution profiles of Triton X-100 insoluble NPM1 and RNA. Endogenous NPM1 was detected by immunoblot. Protein mass standards are indicated above the panel : thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), and Ribonuclease A (13.7 kDa). RNA was detected by ethidium bromide staining. The four NPM1 groups (Group 1 to Group 4) identified by SEC are underlined. ( B ) Schematic representation of sample preparation in C. ( C ) Elution profile of sonication-treated Triton X-100 soluble NPM1 prepared in B.

Article Snippet: Immunoprecipitates were eluted with PBS containing 1% Triton X-100 and 2 M guanidine-HCl (Nacalai Tesque).

Techniques: Staining, Size-exclusion Chromatography, Sample Prep, Sonication

Journal: Scientific Reports

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography

doi: 10.1038/s41598-018-22359-w

Figure Lengend Snippet: Elution profiles of NPM1 monomer and oligomers expressed in cells. 293T cells were transfected with expression vector for G196-NPM1 (WT) or G196-NPM1 (LG). ( A ) The Triton X-100 soluble lysates were used for immunoprecipitation with anti-G196 antibody. G196-NPM1 and extracted RNA were detected by immunoblot and ethidium bromide staining, respectively. ( B ) Interaction of G196-NPM (WT) or G196-NPM1 (LG) with endogenous NPM1. The Triton X-100 soluble (S) and insoluble (I) cell lysates were treated with RNase A, then immunoprecipitated with anti-G196 antibody. ( C ) Elution profiles of G196-NPM1 (WT) in the Triton X-100 soluble and insoluble cell lysates. Fractions containing the four NPM1 groups identified in Fig.  3A are underlined. ( D ) Elution profile of G196-NPM1 (LG) in the Triton X-100-soluble cell lysates. ( E ) Elution profiles of G196-NPM1 (WT) in the RNase A-treated Triton X-100 soluble and insoluble cell lysates. Fractions containing the four NPM1 groups identified in Fig.  3A were underlined.

Article Snippet: Immunoprecipitates were eluted with PBS containing 1% Triton X-100 and 2 M guanidine-HCl (Nacalai Tesque).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Staining