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: bioRxiv

Article Title: Neuraminidase B controls neuraminidase A-dependent mucus production and evasion

doi: 10.1101/2020.12.03.409706

Figure Lengend Snippet: Mucosal carbohydrate-mediated binding of S. pneumoniae to nasal lavages (A-D) Adherence of Spn Type 4 (TIGR4) to murine nasal lavages (mNL) was analyzed in a solid-phase assay. Bacteria (2× 10 4 CFU/100 μl DMEM) were incubated with 100μL of undiluted, immobilized, pooled mNL in presence of 0.1% BSA for 2hr at 30°C. After 19 washes, adherent bacteria were determined by resuspending with 0.001% Triton X-100 following plating on TS agar plates supplemented with 200 μg/ml streptomycin. (A) Prior to immobilization, mNL was sonicated (Amplitude 8μM) for increasing amounts of time followed by blocking with 0.1% BSA and incubation with Spn (B) Filtering of mNL’s with a 0.45uM filter followed by immobilization, blocking with 0.1% BSA and incubation with Spn (C) Treatment of immobilized mNL with 100mM NaIO 4 in 50mM sodium acetate buffer for 30 min at 4°C in the dark followed by blocking with 0.1% BSA and incubation with Spn (D) Treatment of immobilized mNL with 50μg/mL trypsin for 30 min at 37°C followed by the blocking with 0.1% BSA and incubation with Spn. (E-F) Wild-type Spn were incubated with mNL or hNF for 2hr at 37°C and 5% CO 2 . (E) Type 4 (TIGR4) incubated with mNL (F) Type 23F incubated with human nasal fluid (hNF). Mucus (blue) was stained with alcian blue and bacteria (green) were detected using rabbit anti-capsule antibody and secondary FITC-coupled goat anti-rabbit IgG. Spn were visualized by microscopy on an Axiovert 40 CFL microscope equipped with an Axiocam IC digital camera at 100x. Experiments were performed in duplicates and mean values of three independent experiments are shown with error bars corresponding to S.D. *,p

Article Snippet: Triton X-100 (9002-93-1) was obtained from Amresco (Solon, OH, USA).

Techniques: Binding Assay, Incubation, Sonication, Blocking Assay, Staining, Microscopy

Journal: bioRxiv

Article Title: Neuraminidase B controls neuraminidase A-dependent mucus production and evasion

doi: 10.1101/2020.12.03.409706

Figure Lengend Snippet: NanB and NanA mediate mucus evasion Adherence of WT Spn and isogenic mutants to pooled mNL and hNF was assessed in a solid phase assay. Bacteria (2 × 10 4 CFU/ 100 μl DMEM) were incubated with 100μL of undiluted, pooled mNL or 10 μg hNF in presence of 0.1 % BSA for 2hr at 30°C. After 19 washes, adherent bacteria were determined by resuspending with 0.001% Triton X-100 following plating on TS agar plates supplemented with 200 μg/ml streptomycin. (A) Adherence of Type 23F::pilus-1; Type 23F::pilus-1, nanB:: janus and Type 23F::pilus-1, nanB::nanB to mNF (B) Adherence of Type 23F::pilus-1 and Type 23F::pilus-1, nanB:: janus and Type 23F::pilus-1,Δ nanA,nanB ::janus to hNF (C) Adherence of Type 23F::pilus-1 and Type 23F::pilus-1, nanB:: janus and Type 23F::pilus-1,NanB D270A to hNF (D) Adherence of Type 23F::pilus-1; Type 23F::pilus-1,Δ nanA and Type 23F::pilus-1,Δ nanA::nanA to mNF (E) Adherence of Type 23F::pilus-1; Type 23F::pilus-1,Δ nanA and Type 23F::pilus-1,Δ nanA::nanA to hNF. Experiments were performed in duplicates and mean values of three independent experiments are shown with error bars corresponding to S.D. *,p

Article Snippet: Triton X-100 (9002-93-1) was obtained from Amresco (Solon, OH, USA).

Techniques: Incubation

Journal: bioRxiv

Article Title: Neuraminidase B controls neuraminidase A-dependent mucus production and evasion

doi: 10.1101/2020.12.03.409706

Figure Lengend Snippet: Screening of pneumococcal surface factors interacting with mucus Adherence of WT Spn and isogenic mutants to pooled mNL or hNF was assessed in a solid phase assay. Bacteria (2× 10 4 CFU/100μl DMEM) were incubated with 100μL of undiluted, immobilized, pooled mNL in presence of 0.1% BSA for 2hr at 30°C. After 19 washes, adherent bacteria were determined by resuspending with 0.001% Triton X-100 following plating on TS agar plates supplemented with 200 μg/ml streptomycin. (A) Adherence of TIGR4, TIGR4Δ cps and TIGR4Δ cps::cps to mNF. (B) Adherence of TIGR4 and TIGR4Δ rlrA to mNF. (C) Adherence of Type 23F and Type 23F::pilus-1 to hNF. Experiments were performed in duplicates and mean values of three independent experiments are shown with error bars corresponding to S.D. **,p

Article Snippet: Triton X-100 (9002-93-1) was obtained from Amresco (Solon, OH, USA).

Techniques: Incubation

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: 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

Journal: Molecular Biology of the Cell

Article Title: Proteolytic Processing and Ca2+-binding Activity of Dense-Core Vesicle Polypeptides in Tetrahymena

doi:

Figure Lengend Snippet: Calcium titration. Aliquots of isolated granules in 1% Triton X-100 were incubated with the indicated calcium concentrations, followed by addition of chymotrypsin. 60 μg of protein were loaded per lane, and samples were visualized with Coomassie blue. Grl1p is marked by ∗.

Article Snippet: The buffer was 50 mM KCl, 50 mM Tris-HCl pH 9, 1% Triton X-100 (Boehringer Mannheim, Indianapolis, IN), 2.5 mM MgCl2 , 1 μM in each primer, 0.25 mM dNTPs and 2.5U Taq polymerase.

Techniques: Titration, Isolation, Incubation

Journal: Molecular Biology of the Cell

Article Title: Proteolytic Processing and Ca2+-binding Activity of Dense-Core Vesicle Polypeptides in Tetrahymena

doi:

Figure Lengend Snippet: Chymotryptic digestion of Grl1p in isolated granules and cell homogenates. In all three panels, ∗ indicates the position of mature Grl1p. Several of the smaller Grlps cannot be seen on this gel; their chymotrypsin insensitivity was noted in other experiments using higher percentage gels. Lanes 1–5: Aliquots of isolated granules were treated as described in the table beneath the lanes; ∼40 μg of granule protein was loaded per lane of a 15% polyacrylamide gel. In the presence of Triton X-100, chymotrypsin (1 mg/ml) generated distinct fragments of Grl1p (lane 4). Incubation with calcium (1 mM) before addition of chymotrypsin resulted in protection of Grl1p (lane 5). Samples were visualized with Coomassie blue. Lanes 6–12. Isolated granules were incubated with 1% Triton X-100, and aliquots containing 8 μg of protein, were treated as indicated in the table beneath the lanes. After SDS-PAGE, proteins were transferred to nitrocellulose and antibody blotted with antiGrl1p. A ladder of proteolytic products was generated with increasing concentrations of chymotrypsin (lanes 7, 9, 11). At all chymotrypsin concentrations, preincubation with 1 mM calcium provided extensive protection (lanes 8, 10, 12). Lanes 13–16. Aliquots of a crude particulate fraction from MN173 cells were treated as indicated. One hundred and fifty micrograms of total protein was loaded per lane; the chymotrypsin concentration was 0.5 mg/ml. Proteins were visualized by antibody blotting with anti-Grl1p.

Article Snippet: The buffer was 50 mM KCl, 50 mM Tris-HCl pH 9, 1% Triton X-100 (Boehringer Mannheim, Indianapolis, IN), 2.5 mM MgCl2 , 1 μM in each primer, 0.25 mM dNTPs and 2.5U Taq polymerase.

Techniques: Isolation, Generated, Incubation, SDS Page, Concentration Assay