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Roche cacl2
Cacl2, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 97 stars, based on 207 article reviews
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cacl2 - by Bioz Stars, 2020-01
97/100 stars

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Centrifugation:

Article Title: Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons
Article Snippet: Briefly, live transfected HEK293 cells were washed once at 37°C with DPBS with 1 mM CaCl2 and 1 mM MgCl2 , followed by 30 min incubation at 37°C with 10 mM HEPES, 150 mM NaCl, and 2 mM CaCl2 (pH 7.4) with or without 200 μg/mL Proteinase K (Roche). .. Cells were harvested with a cell scraper, pelleted by centrifugation at 4°C at 400 × g for 5 min in a microcentrifuge, and resuspended in reducing SDS sample buffer.

Article Title: Smoothened stimulation by membrane sterols drives Hedgehog pathway activity
Article Snippet: Cells were lysed for 1 h in a buffer containing: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5% LMNG, 0.05% CHS, 100 μM TCEP, 1 mM CaCl2 and complete protease inhibitors (Roche). .. Following centrifugation (20,000g, 4 °C, 20 min), a portion of the clarified lysate was saved as ‘input, and the remainder incubated with M1 Flag affinity resin for 1 h at 4 °C with rotation.

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: .. Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse). .. Unbroken spheroplasts were removed by centrifugation 5,000 rpm for 10 min, and then the membrane fraction was collected by ultracentrifugation at 100,000 g for 1 hr at 4°C.

Article Title: Alzheimer Amyloid-? Oligomer Bound to Post-Synaptic Prion Protein Activates Fyn to Impair Neurons
Article Snippet: Subcellular Fractionation of Brain Tissue Rat forebrains were homogenized in ice-cold 5 mM NaHEPES, pH7.4, 1 mM MgCl2 , 0.5 mM CaCl2 , complete protease inhibitor cocktail (Roche), phosphatase inhibitor (Roche) with a glass/Teflon pestel. .. The P2 suspension was loaded onto a discontinuous sucrose gradient (0.85 M/ 1 M/ 1.15 M sucrose solution in 6 mM TrisHCl, pH 8.0), followed by centrifugation for 2 h at 82,500 × g .

Amplification:

Article Title: Pancreatic β-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes
Article Snippet: Adenovirus was amplified in HEK-293 cells, purified on CsCl gradient, and quantified using the Adeno-X Rapid Titer Kit (Clontech). .. The cells were perifused at a flow rate of ∼1 ml/min with a bicarbonate-buffered Krebs solution containing 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2 , 24 mM NaHCO3 , 1 g/l BSA (Fraction V, Roche) and 10 mM glucose.

Cytometry:

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Purification of human CD34+ cells were assessed by quantifying by flow cytometry using an anti-human CD34+-FITC antibody (dilution 1/100, clone 581, BD Biosciences).

Blocking Assay:

Article Title: The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis
Article Snippet: Whole liver protein extracts were prepared in liver homogenization buffer (50 mmol/L Tris–HCl [pH 7.6], 0.25% Triton-X 100, 0.15 M NaCl, 10 mM CaCl2 and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)). .. Blots were incubated overnight at 4°C in a blocking buffer containing 5% skim milk and then incubated with either anti-αSMA (DAKO, cat no. M0851) or β-actin (Sigma) mouse monoclonal antibody, diluted 1/2000, for 2 h at room temperature, and subsequently, with peroxidase- conjugated goat anti-mouse IgG (PARIS, Compiegne, France) diluted 1/10,000, for 1 h at room temperature.

Article Title: EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase
Article Snippet: The embryo or fishes samples were soaked in a cacodylate-buffered solution containing 2 mM CaCl2 , 30% sucrose, and protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) at 4 ℃ for overnight. .. Then, the samples were quickly frozen in OCT compound (Sakura Finetek, Tokyo, Japan) and 12-μm thickness serial cryostat sections were cut and mounted on glass slides, then quickly blocked by 0.1 M phosphate buffer (pH 7.4) containing 4% Block Ace (DS Pharma Biomedical), protease cocktail, and 0.02% saponin, and incubated at room temperature (RT) for 20 min. Then, the samples were incubated at 4 ℃ for 1 overnight for primary Abs, and the 2 h at RT for secondary Abs.

Incubation:

Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis
Article Snippet: Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)). .. E-selectin-Ig binding proteins were pulled down via incubation with Protein G Agarose beads pre-blocked with BSA, and eluted via boiling in 1.5x reducing sample buffer with SDS.

Article Title: The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis
Article Snippet: Whole liver protein extracts were prepared in liver homogenization buffer (50 mmol/L Tris–HCl [pH 7.6], 0.25% Triton-X 100, 0.15 M NaCl, 10 mM CaCl2 and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)). .. Blots were incubated overnight at 4°C in a blocking buffer containing 5% skim milk and then incubated with either anti-αSMA (DAKO, cat no. M0851) or β-actin (Sigma) mouse monoclonal antibody, diluted 1/2000, for 2 h at room temperature, and subsequently, with peroxidase- conjugated goat anti-mouse IgG (PARIS, Compiegne, France) diluted 1/10,000, for 1 h at room temperature.

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500× g for 5 min. ..

Article Title: Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons
Article Snippet: .. Briefly, live transfected HEK293 cells were washed once at 37°C with DPBS with 1 mM CaCl2 and 1 mM MgCl2 , followed by 30 min incubation at 37°C with 10 mM HEPES, 150 mM NaCl, and 2 mM CaCl2 (pH 7.4) with or without 200 μg/mL Proteinase K (Roche). ..

Article Title: Smoothened stimulation by membrane sterols drives Hedgehog pathway activity
Article Snippet: After 24 h, cells were collected, washed once in Hank’s Balanced Saline Solution, and incubated with DMSO vehicle or the following pharmacological agents at 37 °C for 90 min: KAAD-cyclopamine (1 μM), SAG2lk (1 μM) or MβCD (8 mM). .. Cells were lysed for 1 h in a buffer containing: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5% LMNG, 0.05% CHS, 100 μM TCEP, 1 mM CaCl2 and complete protease inhibitors (Roche).

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Protoplasts were generated by addition of 20 mg/ml lysozyme and 100 units mutanolysin (Sigma) and incubated at 37°C for 30 min. .. Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse).

Article Title: EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase
Article Snippet: The embryo or fishes samples were soaked in a cacodylate-buffered solution containing 2 mM CaCl2 , 30% sucrose, and protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) at 4 ℃ for overnight. .. Then, the samples were quickly frozen in OCT compound (Sakura Finetek, Tokyo, Japan) and 12-μm thickness serial cryostat sections were cut and mounted on glass slides, then quickly blocked by 0.1 M phosphate buffer (pH 7.4) containing 4% Block Ace (DS Pharma Biomedical), protease cocktail, and 0.02% saponin, and incubated at room temperature (RT) for 20 min. Then, the samples were incubated at 4 ℃ for 1 overnight for primary Abs, and the 2 h at RT for secondary Abs.

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: .. Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Human CD34+ HSC were isolated using a CD34+ HSC isolation kit (Stem Cell Technologies) according to the manufacturer' protocol.

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min. ..

Activity Assay:

Article Title: Smoothened stimulation by membrane sterols drives Hedgehog pathway activity
Article Snippet: Paragraph title: SMO activity dependent immunoprecipitation of NbSmo8. ... Cells were lysed for 1 h in a buffer containing: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5% LMNG, 0.05% CHS, 100 μM TCEP, 1 mM CaCl2 and complete protease inhibitors (Roche).

Cell Culture:

Article Title: Role of Multidrug Resistance–Associated Protein 4 in the Basolateral Efflux of Hepatically Derived Enalaprilat
Article Snippet: Paragraph title: Human Embryonic Kidney Cell Culture and Overexpression of MRP3 and MRP4. ... The final cell pellet was overlaid with 10 ml TSB containing 0.25 mM CaCl2 and protease inhibitors (complete Mini EDTA-free; Roche Diagnostics), snap-frozen in liquid nitrogen, and stored at −80°C.

Expressing:

Article Title: Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons
Article Snippet: Proteinase K digestion of HEK293 cells Proteinase K analysis of cell surface expression was performed essentially as described (Zhou et al., ; Manganas and Trimmer, ). .. Briefly, live transfected HEK293 cells were washed once at 37°C with DPBS with 1 mM CaCl2 and 1 mM MgCl2 , followed by 30 min incubation at 37°C with 10 mM HEPES, 150 mM NaCl, and 2 mM CaCl2 (pH 7.4) with or without 200 μg/mL Proteinase K (Roche).

Article Title: Smoothened stimulation by membrane sterols drives Hedgehog pathway activity
Article Snippet: Sodium butyrate was added to 10 mM to enhance expression. .. Cells were lysed for 1 h in a buffer containing: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5% LMNG, 0.05% CHS, 100 μM TCEP, 1 mM CaCl2 and complete protease inhibitors (Roche).

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Expression of human CD90, CD38, CD45RA was assessed among the CD34+ population.

Western Blot:

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500× g for 5 min. ..

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min. ..

Over Expression:

Article Title: Role of Multidrug Resistance–Associated Protein 4 in the Basolateral Efflux of Hepatically Derived Enalaprilat
Article Snippet: Paragraph title: Human Embryonic Kidney Cell Culture and Overexpression of MRP3 and MRP4. ... The final cell pellet was overlaid with 10 ml TSB containing 0.25 mM CaCl2 and protease inhibitors (complete Mini EDTA-free; Roche Diagnostics), snap-frozen in liquid nitrogen, and stored at −80°C.

Flow Cytometry:

Article Title: Pancreatic β-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes
Article Snippet: .. The cells were perifused at a flow rate of ∼1 ml/min with a bicarbonate-buffered Krebs solution containing 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2 , 24 mM NaHCO3 , 1 g/l BSA (Fraction V, Roche) and 10 mM glucose. ..

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Purification of human CD34+ cells were assessed by quantifying by flow cytometry using an anti-human CD34+-FITC antibody (dilution 1/100, clone 581, BD Biosciences).

Protease Inhibitor:

Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis
Article Snippet: .. Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)). .. Cell lysates were precleared by incubating overnight with Protein G agarose beads (Invitrogen) pre-blocked with Bovine Serum Albumin (BSA).

Article Title: The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis
Article Snippet: .. Whole liver protein extracts were prepared in liver homogenization buffer (50 mmol/L Tris–HCl [pH 7.6], 0.25% Triton-X 100, 0.15 M NaCl, 10 mM CaCl2 and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)). ..

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500× g for 5 min. .. For RT-QuIC analysis, the S1 fraction was diluted at 1:1 with 2X conversion buffer containing 300 mM NaCl, 2% Triton X-100 and a complete protease inhibitor in PBS without Ca2+ and Mg2+ to prepare a 5% skin homogenate and then make serial dilution with 0.1% SDS/PBS.

Article Title: EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase
Article Snippet: .. The embryo or fishes samples were soaked in a cacodylate-buffered solution containing 2 mM CaCl2 , 30% sucrose, and protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) at 4 ℃ for overnight. .. Then, the samples were quickly frozen in OCT compound (Sakura Finetek, Tokyo, Japan) and 12-μm thickness serial cryostat sections were cut and mounted on glass slides, then quickly blocked by 0.1 M phosphate buffer (pH 7.4) containing 4% Block Ace (DS Pharma Biomedical), protease cocktail, and 0.02% saponin, and incubated at room temperature (RT) for 20 min. Then, the samples were incubated at 4 ℃ for 1 overnight for primary Abs, and the 2 h at RT for secondary Abs.

Article Title: Alzheimer Amyloid-? Oligomer Bound to Post-Synaptic Prion Protein Activates Fyn to Impair Neurons
Article Snippet: .. Subcellular Fractionation of Brain Tissue Rat forebrains were homogenized in ice-cold 5 mM NaHEPES, pH7.4, 1 mM MgCl2 , 0.5 mM CaCl2 , complete protease inhibitor cocktail (Roche), phosphatase inhibitor (Roche) with a glass/Teflon pestel. ..

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min. .. For RT-QuIC analysis, the S1 fraction was diluted at 1:1 with 2X conversion buffer containing 300 mM NaCl, 2% Triton X-100 and a complete protease inhibitor in PBS without Ca2+ and Mg2+ to prepare a 5% skin homogenate and then make serial dilution with 0.1% SDS/PBS.

Infection:

Article Title: Pancreatic β-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes
Article Snippet: Two days after transfection and 48 h before the fluorescence measurements the cells were infected with adenovirus encoding roGFP2-Orp1. .. The cells were perifused at a flow rate of ∼1 ml/min with a bicarbonate-buffered Krebs solution containing 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2 , 24 mM NaHCO3 , 1 g/l BSA (Fraction V, Roche) and 10 mM glucose.

Article Title: Smoothened stimulation by membrane sterols drives Hedgehog pathway activity
Article Snippet: Suspension HEK293 cells (unauthenticated and untested for mycoplasma contamination; Thermo Fisher) were infected with BacMam viruses encoding an N-terminal streptavidin-binding-peptide-and Flag-tagged SMO truncated at residue 566 and fused to GNAO along with either control Nb80-GFP, which is specific to the β2-adrenergic receptor, or NbSmo8-GFP fusions at a 1:1 ratio of SMO:Nb viruses. .. Cells were lysed for 1 h in a buffer containing: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5% LMNG, 0.05% CHS, 100 μM TCEP, 1 mM CaCl2 and complete protease inhibitors (Roche).

Light Microscopy:

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Complete protoplasting was monitored by light microscopy. .. Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse).

Generated:

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Protoplasts were generated by addition of 20 mg/ml lysozyme and 100 units mutanolysin (Sigma) and incubated at 37°C for 30 min. .. Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse).

Inverted Microscopy:

Article Title: Pancreatic β-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes
Article Snippet: For the dynamic measurements of roGFP2-Orp1, the cell-containing coverslips were mounted in a perifusion chamber maintained at 37°C and placed on a ×40 objective of an inverted microscope. .. The cells were perifused at a flow rate of ∼1 ml/min with a bicarbonate-buffered Krebs solution containing 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2 , 24 mM NaHCO3 , 1 g/l BSA (Fraction V, Roche) and 10 mM glucose.

Binding Assay:

Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis
Article Snippet: Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)). .. E-selectin-Ig binding proteins were pulled down via incubation with Protein G Agarose beads pre-blocked with BSA, and eluted via boiling in 1.5x reducing sample buffer with SDS.

Fluorescence:

Article Title: Pancreatic β-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes
Article Snippet: Two days after transfection and 48 h before the fluorescence measurements the cells were infected with adenovirus encoding roGFP2-Orp1. .. The cells were perifused at a flow rate of ∼1 ml/min with a bicarbonate-buffered Krebs solution containing 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2 , 24 mM NaHCO3 , 1 g/l BSA (Fraction V, Roche) and 10 mM glucose.

Isolation:

Article Title: Altenusin, a Nonsteroidal Microbial Metabolite, Attenuates Nonalcoholic Fatty Liver Disease by Activating the Farnesoid X Receptor
Article Snippet: Paragraph title: Isolation and Culture of Primary Mouse Hepatocytes. ... Briefly, the liver was first perfused with Hanks’ buffered salt solution containing 0.5 mM EGTA and 0.1 M HEPES at 5 ml/min for 5–10 minutes and then perfused with L-15 medium containing 1.8 mM CaCl2 , 0.1 M HEPES, and 20 μg/ml liberase (Roche, Indianapolis, IN).

Article Title: The Na+/Ca2+ exchange inhibitor SEA0400 limits intracellular Ca2+ accumulation and improves recovery of ventricular function when added to cardioplegia
Article Snippet: Paragraph title: Cardioplegic arrest - isolated cardiomyocytes ... Hearts were then perfused with this solution without CaCl2 (5 min), followed by (20 min) buffer containing 50 μM CaCl2 , protease dispase II (0.1 mg/mL, Roche Diagnostics, Laval, Canada), collagenase type 2 (0.56 mg/mL, Worthington, Lakewood, NJ) and trypsin (0.02 mg/mL, Sigma-Aldrich, Oakville, Canada).

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Paragraph title: Isolation and analysis of pneumococcal LTAs ... Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse).

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Paragraph title: Isolation of human CD34+ and murine CD117+ HSC ... Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C.

Transfection:

Article Title: Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons
Article Snippet: .. Briefly, live transfected HEK293 cells were washed once at 37°C with DPBS with 1 mM CaCl2 and 1 mM MgCl2 , followed by 30 min incubation at 37°C with 10 mM HEPES, 150 mM NaCl, and 2 mM CaCl2 (pH 7.4) with or without 200 μg/mL Proteinase K (Roche). ..

Article Title: Pancreatic β-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes
Article Snippet: Two days after transfection and 48 h before the fluorescence measurements the cells were infected with adenovirus encoding roGFP2-Orp1. .. The cells were perifused at a flow rate of ∼1 ml/min with a bicarbonate-buffered Krebs solution containing 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2 , 24 mM NaHCO3 , 1 g/l BSA (Fraction V, Roche) and 10 mM glucose.

Article Title: Role of Multidrug Resistance–Associated Protein 4 in the Basolateral Efflux of Hepatically Derived Enalaprilat
Article Snippet: For MRP3, transient transfection of HEK293T cells with X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostics, Mannheim, Germany) was performed according to the manufacturer’s instructions. .. The final cell pellet was overlaid with 10 ml TSB containing 0.25 mM CaCl2 and protease inhibitors (complete Mini EDTA-free; Roche Diagnostics), snap-frozen in liquid nitrogen, and stored at −80°C.

Purification:

Article Title: Pancreatic β-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes
Article Snippet: Adenovirus was amplified in HEK-293 cells, purified on CsCl gradient, and quantified using the Adeno-X Rapid Titer Kit (Clontech). .. The cells were perifused at a flow rate of ∼1 ml/min with a bicarbonate-buffered Krebs solution containing 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2 , 24 mM NaHCO3 , 1 g/l BSA (Fraction V, Roche) and 10 mM glucose.

Article Title: Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome
Article Snippet: Fetal liver was homogenized and incubated in digestion medium (HBSS with 0.1% collagenase IV (Sigma), 40 mM HEPES, 2 M CaCl2 and 2 U ml−1 DNAse I (Roche) for 30 min at 37 °C. .. Purification of human CD34+ cells were assessed by quantifying by flow cytometry using an anti-human CD34+-FITC antibody (dilution 1/100, clone 581, BD Biosciences).

Polyacrylamide Gel Electrophoresis:

Article Title: A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis
Article Snippet: Protoplasts were pelleted by centrifugation at 5000 g for 5 min and resuspended in 2 ml cold hypotonic buffer to lyse them (20 mM HEPES (Na+ ), pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2 , 1 mM CaCl2 , 2X complete protease inhibitors (Roche), 6 μg/ml RNAse A, 6 μg/ml DNAse). .. The pellet was resuspended in 1 ml SDS sample buffer (200 mM Tris-HCL, pH 6.8, 40% glycerol, 2% SDS, 0.04% Coomassie Blue G-250), boiled for 10 min, and separated by Tris-tricine PAGE followed by immunoblotting with anti-PCho monoclonal antibody TEPC-15 (Sigma).

Mouse Assay:

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: Skin samples at 0.5 × 0.5 cm from ear pinna, thigh, back, belly, or rib were collected from hamsters inoculated with 263K scrapie, Tg40h mice, or PBS-negative controls, respectively. .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500× g for 5 min.

Article Title: Altenusin, a Nonsteroidal Microbial Metabolite, Attenuates Nonalcoholic Fatty Liver Disease by Activating the Farnesoid X Receptor
Article Snippet: Primary mouse hepatocytes were isolated from 6- to 8-week-old male mice, as previously described ( ). .. Briefly, the liver was first perfused with Hanks’ buffered salt solution containing 0.5 mM EGTA and 0.1 M HEPES at 5 ml/min for 5–10 minutes and then perfused with L-15 medium containing 1.8 mM CaCl2 , 0.1 M HEPES, and 20 μg/ml liberase (Roche, Indianapolis, IN).

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: Skin samples at 0.5 × 0.5 cm from ear pinna, thigh, back, belly, or rib were collected from hamsters inoculated with 263K scrapie, Tg40h mice, or PBS-negative controls, respectively. .. For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min.

In Situ:

Article Title: The Na+/Ca2+ exchange inhibitor SEA0400 limits intracellular Ca2+ accumulation and improves recovery of ventricular function when added to cardioplegia
Article Snippet: Briefly, hearts were perfused in situ for 5 min with buffer containing (mM): 135.5 NaCl, 4 KCl, 10 HEPES, 1.2 MgSO4 , 1.2 KH2 PO4 12 glucose and 200 μM CaCl2 (pH 7.4, 37°C, 100% O2 ). .. Hearts were then perfused with this solution without CaCl2 (5 min), followed by (20 min) buffer containing 50 μM CaCl2 , protease dispase II (0.1 mg/mL, Roche Diagnostics, Laval, Canada), collagenase type 2 (0.56 mg/mL, Worthington, Lakewood, NJ) and trypsin (0.02 mg/mL, Sigma-Aldrich, Oakville, Canada).

Homogenization:

Article Title: Prion Seeds Distribute throughout the Eyes of Sporadic Creutzfeldt-Jakob Disease Patients
Article Snippet: Eye homogenates (10% [wt/vol]) were prepared in PBS containing 2 mM CaCl2 and 0.25% (wt/vol) collagenase A (Roche). .. Glass homogenization beads (1 mm; Biospec) were added to the sample in homogenization buffer and tissues were homogenized for 1 min using a Beadbeater tissue homogenizer (Biospec).

Article Title: The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis
Article Snippet: .. Whole liver protein extracts were prepared in liver homogenization buffer (50 mmol/L Tris–HCl [pH 7.6], 0.25% Triton-X 100, 0.15 M NaCl, 10 mM CaCl2 and complete mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)). ..

Immunoprecipitation:

Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis
Article Snippet: Paragraph title: Immunoprecipitation with E-selectin-IgG ... Cell lysates were prepared in Selectin wash/lysis buffer (2%NP40, 150mM NaCl, 50mM Tris-HCl (pH7.4), 2mM CaCl2 , 20μg/mL PMSF, and 1x protease inhibitor cocktail (Roche)).

Article Title: Smoothened stimulation by membrane sterols drives Hedgehog pathway activity
Article Snippet: Paragraph title: SMO activity dependent immunoprecipitation of NbSmo8. ... Cells were lysed for 1 h in a buffer containing: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5% LMNG, 0.05% CHS, 100 μM TCEP, 1 mM CaCl2 and complete protease inhibitors (Roche).

Fractionation:

Article Title: Alzheimer Amyloid-? Oligomer Bound to Post-Synaptic Prion Protein Activates Fyn to Impair Neurons
Article Snippet: .. Subcellular Fractionation of Brain Tissue Rat forebrains were homogenized in ice-cold 5 mM NaHEPES, pH7.4, 1 mM MgCl2 , 0.5 mM CaCl2 , complete protease inhibitor cocktail (Roche), phosphatase inhibitor (Roche) with a glass/Teflon pestel. ..

Serial Dilution:

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500× g for 5 min. .. For RT-QuIC analysis, the S1 fraction was diluted at 1:1 with 2X conversion buffer containing 300 mM NaCl, 2% Triton X-100 and a complete protease inhibitor in PBS without Ca2+ and Mg2+ to prepare a 5% skin homogenate and then make serial dilution with 0.1% SDS/PBS.

Article Title: Early preclinical detection of prions in the skin of prion-infected animals
Article Snippet: For western blotting, or sPMCA assay, the skin samples were incubated in TBS containing 2 mM CaCl2 and 0.25% (10% w/v) collagenase A (Roche) in a shaker at 37 °C for 4 h. Beads Beater was used to make tissue homogenates and the samples were then centrifuged at 500×g for 5 min. .. For RT-QuIC analysis, the S1 fraction was diluted at 1:1 with 2X conversion buffer containing 300 mM NaCl, 2% Triton X-100 and a complete protease inhibitor in PBS without Ca2+ and Mg2+ to prepare a 5% skin homogenate and then make serial dilution with 0.1% SDS/PBS.

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    Roche annexin v binding buffer
    TRAIL plus SB203580 activates a secondary JNK pathway that upregulates MCL-1 expression and partially protects cells from apoptosis. ( a ) DU145 cells were treated with TRAIL (500 ng/ml) ±SB203580 (50 μM) and immunoblotted for active p-JNK (Thr-183/Tyr-185), p-ERK (Thr-202/Tyr-204), and p-AKT (Ser-473). ( b ) DU145 and LNCaP prostate cancer cells treated with TRAIL (500 ng/ml) ± SB203580 (50 μM) and the activity of JNKs determined by immunoblotting for phospho-c-Jun (Ser-73). ( c–e ) DU145 cells were treated with TRAIL±SB203580± JIP  peptide (10 μM) and assayed for MCL-1 expression at the protein (immunoblotting) and transcriptional (qRT-PCR) levels. Apoptosis was determined by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. Note that in the inset to ( e ), the inhibitory effect of JIP was confirmed, as it inhibited JNK-dependent phosphorylation of c-Jun
    Annexin V Binding Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Roche regular tyrode solution
    Voltage-gated sodium current ( I Na ) in isolated basal neurones endowed with an axon stalk A , differential interference contrast (Nomarski optics) photomicrograph (left) of a basal neurone isolated from mouse VNO. Note the axon stalk (a) emerging from the soma. Dendrite (d) length: 50 μm. Patch-clamp recording from this neurone (right) revealed the presence of small-amplitude, voltage-gated Na + currents (downward deflection in the current records). The cell was held at -84 mV and stepped in 10 mV increments from -74 mV to +46 mV. Pipette solution: <t>KCl.</t> Bath solution: <t>Tyrode</t> solution. Scale bar: 10 μm. B , current-voltage relationship for I Na recorded from 11 basal neurones (dendrite length range: 46-89 μm). I Na values for each membrane potential ( V m ) were averaged. Pipette solution: KCl. Bath solution: Tyrode solution.
    Regular Tyrode Solution, supplied by Roche, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Roche annexin buffer
    PP2A is involved in VT-1-induced apoptosis. (A) Ramos cells were incubated with okadaic acid (50 nM) or with vehicle (DMSO) or without any treatment (−) for 1 h before being treated with VT-1 for 4 h. The cells were labeled with <t>annexin</t> V-FITC and PI and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. (B) VT-1-sensitive or -resistant cells were incubated with or without VT-1 (5 ng/ml) for various periods of time and lysed. The lysates were immunoprecipitated with an anti-PP2A Ab, and the levels of PP2A activity were determined by measuring the release of phosphate from a phosphopeptide substrate in a colorimetric assay. The increases in the PP2A activity levels of treated samples were determined with respect to the levels in untreated samples. Error bars indicate the standard deviations.
    Annexin Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 82/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Roche dnase i digestion buffer
    Quantifying changes in DNase I-sensitivity with qrt-PCR. qrt-PCR was used to detected small changes in DNase I-sensitivity in two reciprocally expressed genes ( redD and rlpA ) at the earliest and latest time points. In vivo digestions were performed as described in Figure 1 ; qrt-PCR is described elsewhere ( 6 ). <t>DNase</t> I sensitivity was estimated by the percentage loss of copy number after the rate of digestion had reached a steady level (commonly after 4 U of enzyme had been used).
    Dnase I Digestion Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TRAIL plus SB203580 activates a secondary JNK pathway that upregulates MCL-1 expression and partially protects cells from apoptosis. ( a ) DU145 cells were treated with TRAIL (500 ng/ml) ±SB203580 (50 μM) and immunoblotted for active p-JNK (Thr-183/Tyr-185), p-ERK (Thr-202/Tyr-204), and p-AKT (Ser-473). ( b ) DU145 and LNCaP prostate cancer cells treated with TRAIL (500 ng/ml) ± SB203580 (50 μM) and the activity of JNKs determined by immunoblotting for phospho-c-Jun (Ser-73). ( c–e ) DU145 cells were treated with TRAIL±SB203580± JIP  peptide (10 μM) and assayed for MCL-1 expression at the protein (immunoblotting) and transcriptional (qRT-PCR) levels. Apoptosis was determined by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. Note that in the inset to ( e ), the inhibitory effect of JIP was confirmed, as it inhibited JNK-dependent phosphorylation of c-Jun

    Journal: Cell death and differentiation

    Article Title: TRAIL-activated stress kinases suppress apoptosis through transcriptional upregulation of MCL-1

    doi: 10.1038/cdd.2010.9

    Figure Lengend Snippet: TRAIL plus SB203580 activates a secondary JNK pathway that upregulates MCL-1 expression and partially protects cells from apoptosis. ( a ) DU145 cells were treated with TRAIL (500 ng/ml) ±SB203580 (50 μM) and immunoblotted for active p-JNK (Thr-183/Tyr-185), p-ERK (Thr-202/Tyr-204), and p-AKT (Ser-473). ( b ) DU145 and LNCaP prostate cancer cells treated with TRAIL (500 ng/ml) ± SB203580 (50 μM) and the activity of JNKs determined by immunoblotting for phospho-c-Jun (Ser-73). ( c–e ) DU145 cells were treated with TRAIL±SB203580± JIP peptide (10 μM) and assayed for MCL-1 expression at the protein (immunoblotting) and transcriptional (qRT-PCR) levels. Apoptosis was determined by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. Note that in the inset to ( e ), the inhibitory effect of JIP was confirmed, as it inhibited JNK-dependent phosphorylation of c-Jun

    Article Snippet: Cells were harvested by trypsinization, washed with PBS, and resuspended in annexin-V binding buffer (10mM HEPES, pH 7.4, 140 mM NaCl, 2.5mM CaCl2 ) containing annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

    p38 MAPKs suppress TRAIL-induced apoptosis downstream of the DISC by inhibiting MOMP and the release of Smac/DIABLO. ( a ) Scheme of the antiapoptotic TAK1→MKK3/MKK6→p38 MAPK and proapoptotic FADD→caspase-8→caspase-3 pathways initiated by TRAIL. ( b ) DU145 cells were treated with biotinylated-TRAIL (bTRAIL; 500 ng/ml) ±SB203580 (50 μM), and DISC analyses were performed as described in the Materials and Methods. Note that the unstimulated control (U/S) was obtained by adding bTRAIL to lysed control cells to rule out nonspecific ligand interactions, and the pulldown of endogenously biotinylated acetyl-CoA carboxylase (ACC) was used as a loading control. ( c ) Cells were treated with TRAIL±SB203580±z-VAD-fmk for 0.5–2 h and immunoblotted for c-FLIP. ( d ) Cells were treated with TRAIL±SB203580, and mitochondrial pellets and supernatants were immunoblotted for cytochrome  c  or Smac/DIABLO. Cells were also assayed for changes in Δψ m  by flow cytometry using the fluorescent dye TMRE (25 nM). ( e, f ) DU145 cells, infected with empty virus or virus expressing a dominant-negative caspase-9 (C287A), were treated with TRAIL±SB203580 and assayed for cell death by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. To confirm the effectiveness of the dominant-negative caspase-9, lysates from untreated cells were assayed for apoptosome activity in response to the addition of cytochrome  c  and dATP. ( g   (see inset for expression of cytosolic Smac/DIABLO). Cells were then exposed to TRAIL±SB203580 for 8 h, stained with Hoechst 33258, and assayed for the percentage of GFP+ apoptotic cells. Each bar represents the mean of three separate experiments±S.E.M. NS, not statistically significant. ( h ) DU145 cells were treated with TRAIL±SB203580 and immunoblotted for changes in XIAP expression over a time course (0.5–8 h)

    Journal: Cell death and differentiation

    Article Title: TRAIL-activated stress kinases suppress apoptosis through transcriptional upregulation of MCL-1

    doi: 10.1038/cdd.2010.9

    Figure Lengend Snippet: p38 MAPKs suppress TRAIL-induced apoptosis downstream of the DISC by inhibiting MOMP and the release of Smac/DIABLO. ( a ) Scheme of the antiapoptotic TAK1→MKK3/MKK6→p38 MAPK and proapoptotic FADD→caspase-8→caspase-3 pathways initiated by TRAIL. ( b ) DU145 cells were treated with biotinylated-TRAIL (bTRAIL; 500 ng/ml) ±SB203580 (50 μM), and DISC analyses were performed as described in the Materials and Methods. Note that the unstimulated control (U/S) was obtained by adding bTRAIL to lysed control cells to rule out nonspecific ligand interactions, and the pulldown of endogenously biotinylated acetyl-CoA carboxylase (ACC) was used as a loading control. ( c ) Cells were treated with TRAIL±SB203580±z-VAD-fmk for 0.5–2 h and immunoblotted for c-FLIP. ( d ) Cells were treated with TRAIL±SB203580, and mitochondrial pellets and supernatants were immunoblotted for cytochrome c or Smac/DIABLO. Cells were also assayed for changes in Δψ m by flow cytometry using the fluorescent dye TMRE (25 nM). ( e, f ) DU145 cells, infected with empty virus or virus expressing a dominant-negative caspase-9 (C287A), were treated with TRAIL±SB203580 and assayed for cell death by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. To confirm the effectiveness of the dominant-negative caspase-9, lysates from untreated cells were assayed for apoptosome activity in response to the addition of cytochrome c and dATP. ( g (see inset for expression of cytosolic Smac/DIABLO). Cells were then exposed to TRAIL±SB203580 for 8 h, stained with Hoechst 33258, and assayed for the percentage of GFP+ apoptotic cells. Each bar represents the mean of three separate experiments±S.E.M. NS, not statistically significant. ( h ) DU145 cells were treated with TRAIL±SB203580 and immunoblotted for changes in XIAP expression over a time course (0.5–8 h)

    Article Snippet: Cells were harvested by trypsinization, washed with PBS, and resuspended in annexin-V binding buffer (10mM HEPES, pH 7.4, 140 mM NaCl, 2.5mM CaCl2 ) containing annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Flow Cytometry, Cytometry, Infection, Expressing, Dominant Negative Mutation, Staining, Activity Assay

    TRAIL stimulates BID cleavage, but p38 inhibition is essential to suppress MCL-1 expression, facilitate BAK activation and MOMP, and induce apoptosis. ( a, b ) DU145 cells were treated with TRAIL (500 ng/ml) ±SB203580 (50 μM) ±z-VAD-fmk (50 μM), immunoblotted for BID cleavage, and assayed for BAK activation by flow cytometry using an epitope-specific antibody that recognizes its active conformation. The cells in ( b ) were stably transfected with a scrambled shRNA plasmid and served as controls for ( k ); identical results were obtained in untransfected cells. ( c, d ) Cells were treated with TRAIL±SB203580 and assayed for expression of antiapoptotic multidomain BCL-2 family members, MCL-1, BCL-2, and BCL-x L , at the protein (immunoblotting) and/or transcriptional (qRT-PCR) levels. ( e, f ) Time course experiments were performed (as described above) to assess temporal changes in MCL-1 expression after treatment with TRAIL±SB203580. ( g ) DU145 cells were transiently transfected with pcDNA3-MCL-1 for 24 h, after which cycloheximide (CHX; 1 μM) was added, along with either SB203580 (50 μM) or the ERK inhibitor U0126 (10 μM). Turnover was then assessed by immunoblotting MCL-1 over a 3-h time course. ( h ) DU145 cells were transiently transfected with pEGFP or pEGFP-MCL-1 for 24 h (see inset for immunoblot of expressed proteins), exposed to TRAIL±SB203580 for 8 h, stained with Hoechst 33258, and assayed for the percentage of GFP+ apoptotic cells. Each bar represents the mean of three separate experiments±S.E.M. ( i ) DU145 cells were transfected with pSuper-scramble or pSuper-MCL-1 to stably knockdown the expression of MCL-1 by RNAi (see inset for knockdown of MCL-1). Cells were then exposed to TRAIL±SB203580 and assayed for cell death by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. ( j ) DU145 cells were transiently transfected with pmCherry, pmCherry-NOXA, or pmCherry-BAD for 24 h (see inset for immunoblot of expressed proteins), exposed to TRAIL±SB203580 for 8 h, stained with Hoechst 33258, and assayed for the percentage of mCherry+ apoptotic cells. Each bar represents the mean of three separate experiments±S.E.M. ( k ) DU145 cells were depleted of MCL-1 by RNAi and assayed for BAK activation after treatment with TRAIL±SB203580, as described in ( b ). ( l ) DU145 cells were transiently transfected with EGFP or tBID-EGFP for 24 h (see inset for immunoblot of expressed proteins), exposed to DMSO or SB203580 for 8 h, stained with Hoechst 33258, and assayed for the percentage of GFP+ apoptotic cells. Each bar represents the mean of three separate experiments±S.E.M.

    Journal: Cell death and differentiation

    Article Title: TRAIL-activated stress kinases suppress apoptosis through transcriptional upregulation of MCL-1

    doi: 10.1038/cdd.2010.9

    Figure Lengend Snippet: TRAIL stimulates BID cleavage, but p38 inhibition is essential to suppress MCL-1 expression, facilitate BAK activation and MOMP, and induce apoptosis. ( a, b ) DU145 cells were treated with TRAIL (500 ng/ml) ±SB203580 (50 μM) ±z-VAD-fmk (50 μM), immunoblotted for BID cleavage, and assayed for BAK activation by flow cytometry using an epitope-specific antibody that recognizes its active conformation. The cells in ( b ) were stably transfected with a scrambled shRNA plasmid and served as controls for ( k ); identical results were obtained in untransfected cells. ( c, d ) Cells were treated with TRAIL±SB203580 and assayed for expression of antiapoptotic multidomain BCL-2 family members, MCL-1, BCL-2, and BCL-x L , at the protein (immunoblotting) and/or transcriptional (qRT-PCR) levels. ( e, f ) Time course experiments were performed (as described above) to assess temporal changes in MCL-1 expression after treatment with TRAIL±SB203580. ( g ) DU145 cells were transiently transfected with pcDNA3-MCL-1 for 24 h, after which cycloheximide (CHX; 1 μM) was added, along with either SB203580 (50 μM) or the ERK inhibitor U0126 (10 μM). Turnover was then assessed by immunoblotting MCL-1 over a 3-h time course. ( h ) DU145 cells were transiently transfected with pEGFP or pEGFP-MCL-1 for 24 h (see inset for immunoblot of expressed proteins), exposed to TRAIL±SB203580 for 8 h, stained with Hoechst 33258, and assayed for the percentage of GFP+ apoptotic cells. Each bar represents the mean of three separate experiments±S.E.M. ( i ) DU145 cells were transfected with pSuper-scramble or pSuper-MCL-1 to stably knockdown the expression of MCL-1 by RNAi (see inset for knockdown of MCL-1). Cells were then exposed to TRAIL±SB203580 and assayed for cell death by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. ( j ) DU145 cells were transiently transfected with pmCherry, pmCherry-NOXA, or pmCherry-BAD for 24 h (see inset for immunoblot of expressed proteins), exposed to TRAIL±SB203580 for 8 h, stained with Hoechst 33258, and assayed for the percentage of mCherry+ apoptotic cells. Each bar represents the mean of three separate experiments±S.E.M. ( k ) DU145 cells were depleted of MCL-1 by RNAi and assayed for BAK activation after treatment with TRAIL±SB203580, as described in ( b ). ( l ) DU145 cells were transiently transfected with EGFP or tBID-EGFP for 24 h (see inset for immunoblot of expressed proteins), exposed to DMSO or SB203580 for 8 h, stained with Hoechst 33258, and assayed for the percentage of GFP+ apoptotic cells. Each bar represents the mean of three separate experiments±S.E.M.

    Article Snippet: Cells were harvested by trypsinization, washed with PBS, and resuspended in annexin-V binding buffer (10mM HEPES, pH 7.4, 140 mM NaCl, 2.5mM CaCl2 ) containing annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Inhibition, Expressing, Activation Assay, Flow Cytometry, Cytometry, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Quantitative RT-PCR, Staining

    Inhibition of p38 MAPKs potentiates TRAIL-induced apoptosis in androgen receptor-positive and BAX-expressing prostate cancer cells. ( a ) BAX-deficient DU145 cells, and those stably expressing BAX (see inset for immunoblot of expressed proteins), were treated with TRAIL (500 ng/ml)±SB203580 (50 μM) and assayed for cell death by annexin V/PI staining and flow cytometry. ( b, c   were similarly immunoblotted for MCL-1 and assayed for their sensitivity to TRAIL±SB203580

    Journal: Cell death and differentiation

    Article Title: TRAIL-activated stress kinases suppress apoptosis through transcriptional upregulation of MCL-1

    doi: 10.1038/cdd.2010.9

    Figure Lengend Snippet: Inhibition of p38 MAPKs potentiates TRAIL-induced apoptosis in androgen receptor-positive and BAX-expressing prostate cancer cells. ( a ) BAX-deficient DU145 cells, and those stably expressing BAX (see inset for immunoblot of expressed proteins), were treated with TRAIL (500 ng/ml)±SB203580 (50 μM) and assayed for cell death by annexin V/PI staining and flow cytometry. ( b, c were similarly immunoblotted for MCL-1 and assayed for their sensitivity to TRAIL±SB203580

    Article Snippet: Cells were harvested by trypsinization, washed with PBS, and resuspended in annexin-V binding buffer (10mM HEPES, pH 7.4, 140 mM NaCl, 2.5mM CaCl2 ) containing annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Inhibition, Expressing, Stable Transfection, Staining, Flow Cytometry, Cytometry

    TRAIL activates a TAK1→MKK3/MKK6→p38 MAPK pathway that suppresses apoptosis. ( a ) DU145 prostate cancer cells were treated with recombinant TRAIL (500 ng/ml) for 0.5–8 h, and cells were immunoblotted for various active phosphorylated kinases, including p-p38α (Thr-180/Tyr-182), p-MK2 (Thr-222), p-JNK (Thr-183/Tyr-185), and p-ERK (Thr-202/Tyr-204). ( b, c ) Cells were treated with TRAIL±SB203580 (50 μM)±z-VAD-fmk (50 μM) and assayed for cell death by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. Cells were also immunoblotted for phosphorylated p38, as well as active caspase-8 (p18 large subunit) and caspase-3 (p20, p19, and p17 large subunits). Caspase-3/7 DEVDase activity was also measured, as described in the Materials and Methods. ( d, e ) DU145 cells were transiently transfected with expression plasmids encoding EGFP, constitutively active (D176A/F327S) p38-α (EGFP-p38α-CA), or p38α-CA containing a T106M `gate-keeper' mutation (EGFP-p38α-CA (T106M)). Cells were then exposed to TRAIL for 8 h, stained with Hoechst 33258, and assayed for apoptosis by measuring the percentage of GFP+ cells with condensed nuclei using fluorescence microscopy. Each bar represents the mean of three separate experiments±S.E.M. Note that EGFP-p38α-CA undergoes autophosphorylation and phosphorylates endogenous p38-α, but is sensitive to SB203580, whereas EGFP-p38α-CA (T106M) is only weakly inhibited by SB203580. ( f    ( g, h ) Cells were cotreated with TRAIL±5Z-7-oxozeaenol (5Z-7-oxo; 1 μM)±z-VAD-fmk (50 μM) and were blotted for phosphorylated p-TAK1 (Thr-184/Thr-187) and p-p38 (Thr-180/Tyr-182). The asterisk denotes a nonspecific band. In addition, cells were treated with TRAIL±5Z-7-oxozeaenol or SB203580 and were assayed for cell death by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. ( i ) DU145 cells were transiently transfected with pEGFP for 24 h, along with empty vectors (pcDNA3-FLAG; pcDNA3-HA; and pcDNA6-Myc), or those expressing dominant-negative mutants of TAK1 (K63A), MKK3 (S189A/T193A), MKK6 (S207A/T211A), or p38-α (D168A). Cells were then exposed to TRAIL for 8 h, stained with Hoechst 33258, and assayed for apoptosis by measuring the percentage of GFP+ cells with condensed nuclei. Each bar represents the mean of three separate experiments±S.E.M. Cell lysates were also immunoblotted for FLAG-TAK1-DN, HA-MKK3-DN, HA-MKK6-DN, Myc-p38-DN, and endogenous p-p38α

    Journal: Cell death and differentiation

    Article Title: TRAIL-activated stress kinases suppress apoptosis through transcriptional upregulation of MCL-1

    doi: 10.1038/cdd.2010.9

    Figure Lengend Snippet: TRAIL activates a TAK1→MKK3/MKK6→p38 MAPK pathway that suppresses apoptosis. ( a ) DU145 prostate cancer cells were treated with recombinant TRAIL (500 ng/ml) for 0.5–8 h, and cells were immunoblotted for various active phosphorylated kinases, including p-p38α (Thr-180/Tyr-182), p-MK2 (Thr-222), p-JNK (Thr-183/Tyr-185), and p-ERK (Thr-202/Tyr-204). ( b, c ) Cells were treated with TRAIL±SB203580 (50 μM)±z-VAD-fmk (50 μM) and assayed for cell death by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. Cells were also immunoblotted for phosphorylated p38, as well as active caspase-8 (p18 large subunit) and caspase-3 (p20, p19, and p17 large subunits). Caspase-3/7 DEVDase activity was also measured, as described in the Materials and Methods. ( d, e ) DU145 cells were transiently transfected with expression plasmids encoding EGFP, constitutively active (D176A/F327S) p38-α (EGFP-p38α-CA), or p38α-CA containing a T106M `gate-keeper' mutation (EGFP-p38α-CA (T106M)). Cells were then exposed to TRAIL for 8 h, stained with Hoechst 33258, and assayed for apoptosis by measuring the percentage of GFP+ cells with condensed nuclei using fluorescence microscopy. Each bar represents the mean of three separate experiments±S.E.M. Note that EGFP-p38α-CA undergoes autophosphorylation and phosphorylates endogenous p38-α, but is sensitive to SB203580, whereas EGFP-p38α-CA (T106M) is only weakly inhibited by SB203580. ( f ( g, h ) Cells were cotreated with TRAIL±5Z-7-oxozeaenol (5Z-7-oxo; 1 μM)±z-VAD-fmk (50 μM) and were blotted for phosphorylated p-TAK1 (Thr-184/Thr-187) and p-p38 (Thr-180/Tyr-182). The asterisk denotes a nonspecific band. In addition, cells were treated with TRAIL±5Z-7-oxozeaenol or SB203580 and were assayed for cell death by annexin V/PI staining and flow cytometry. Each bar represents the mean of three separate experiments±S.E.M. ( i ) DU145 cells were transiently transfected with pEGFP for 24 h, along with empty vectors (pcDNA3-FLAG; pcDNA3-HA; and pcDNA6-Myc), or those expressing dominant-negative mutants of TAK1 (K63A), MKK3 (S189A/T193A), MKK6 (S207A/T211A), or p38-α (D168A). Cells were then exposed to TRAIL for 8 h, stained with Hoechst 33258, and assayed for apoptosis by measuring the percentage of GFP+ cells with condensed nuclei. Each bar represents the mean of three separate experiments±S.E.M. Cell lysates were also immunoblotted for FLAG-TAK1-DN, HA-MKK3-DN, HA-MKK6-DN, Myc-p38-DN, and endogenous p-p38α

    Article Snippet: Cells were harvested by trypsinization, washed with PBS, and resuspended in annexin-V binding buffer (10mM HEPES, pH 7.4, 140 mM NaCl, 2.5mM CaCl2 ) containing annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; Roche Applied Sciences, Indianapolis, IN, USA).

    Techniques: Recombinant, Staining, Flow Cytometry, Cytometry, Activity Assay, Transfection, Expressing, Mutagenesis, Fluorescence, Microscopy, Dominant Negative Mutation

    Voltage-gated sodium current ( I Na ) in isolated basal neurones endowed with an axon stalk A , differential interference contrast (Nomarski optics) photomicrograph (left) of a basal neurone isolated from mouse VNO. Note the axon stalk (a) emerging from the soma. Dendrite (d) length: 50 μm. Patch-clamp recording from this neurone (right) revealed the presence of small-amplitude, voltage-gated Na + currents (downward deflection in the current records). The cell was held at -84 mV and stepped in 10 mV increments from -74 mV to +46 mV. Pipette solution: KCl. Bath solution: Tyrode solution. Scale bar: 10 μm. B , current-voltage relationship for I Na recorded from 11 basal neurones (dendrite length range: 46-89 μm). I Na values for each membrane potential ( V m ) were averaged. Pipette solution: KCl. Bath solution: Tyrode solution.

    Journal: The Journal of Physiology

    Article Title: Apical and basal neurones isolated from the mouse vomeronasal organ differ for voltage-dependent currents

    doi: 10.1113/jphysiol.2003.052035

    Figure Lengend Snippet: Voltage-gated sodium current ( I Na ) in isolated basal neurones endowed with an axon stalk A , differential interference contrast (Nomarski optics) photomicrograph (left) of a basal neurone isolated from mouse VNO. Note the axon stalk (a) emerging from the soma. Dendrite (d) length: 50 μm. Patch-clamp recording from this neurone (right) revealed the presence of small-amplitude, voltage-gated Na + currents (downward deflection in the current records). The cell was held at -84 mV and stepped in 10 mV increments from -74 mV to +46 mV. Pipette solution: KCl. Bath solution: Tyrode solution. Scale bar: 10 μm. B , current-voltage relationship for I Na recorded from 11 basal neurones (dendrite length range: 46-89 μm). I Na values for each membrane potential ( V m ) were averaged. Pipette solution: KCl. Bath solution: Tyrode solution.

    Article Snippet: After centrifugation, the supernatant was replaced with regular Tyrode solution (mM: 140 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 , 10 Hepes, 10 glucose, 10 sodium pyruvate, pH adjusted to 7.4 with NaOH) supplemented with DNase (0.1 mg ml−1 ; type I, Roche Diagnostics, Milano, Italy) and incubated for 15 min with agitation.

    Techniques: Isolation, Patch Clamp, Transferring

    Biophysical and pharmacological properties of voltage-gated K + currents ( I K ) in apical and basal neurones A , current-voltage relationships. Amplitude values of I K for each membrane potential ( V m ) were averaged within each group (45 apical neurones ○, 41 basal neurones •). I K amplitude was measured at the end of 40 ms pulses. I K activated at approximately -40 mV. B , inactivation of I K . Currents were elicited by a 400 ms depolarizing step to +46 mV from a holding potential of -84 mV (top). Note the decrease in I K amplitude during prolonged voltage pulses (inactivation). Inactivation properties of I K were evaluated by measuring the ratio between the current peak and the current amplitude at 400 ms. The histograms represent mean values ± S.E.M. from 14-15 measurements of this ratio. Dendrite length range in these experiments: apical neurones, 11-35 μm; basal neurones, 52-104 μm. C , TEA sensitivity of I K recorded in apical and basal neurones. K + current was elicited by a 40 ms test pulse to +46 mV from a holding potential of -84 mV (top). Percentage inhibition of the current amplitude measured at 40 ms was evaluated for 3.34 mM TEA, which is the IC 50 ). In this apical neurone, the reduction was 63 %, whereas in the basal one it was 58 %. The histograms represent mean values ± S.E.M. from 11-13 measurements. Pipette solution: KCl. Bath solution: Tyrode solution. Dendrite length range: apical neurones, 8-28 μm; basal neurones, 50-79 μm.

    Journal: The Journal of Physiology

    Article Title: Apical and basal neurones isolated from the mouse vomeronasal organ differ for voltage-dependent currents

    doi: 10.1113/jphysiol.2003.052035

    Figure Lengend Snippet: Biophysical and pharmacological properties of voltage-gated K + currents ( I K ) in apical and basal neurones A , current-voltage relationships. Amplitude values of I K for each membrane potential ( V m ) were averaged within each group (45 apical neurones ○, 41 basal neurones •). I K amplitude was measured at the end of 40 ms pulses. I K activated at approximately -40 mV. B , inactivation of I K . Currents were elicited by a 400 ms depolarizing step to +46 mV from a holding potential of -84 mV (top). Note the decrease in I K amplitude during prolonged voltage pulses (inactivation). Inactivation properties of I K were evaluated by measuring the ratio between the current peak and the current amplitude at 400 ms. The histograms represent mean values ± S.E.M. from 14-15 measurements of this ratio. Dendrite length range in these experiments: apical neurones, 11-35 μm; basal neurones, 52-104 μm. C , TEA sensitivity of I K recorded in apical and basal neurones. K + current was elicited by a 40 ms test pulse to +46 mV from a holding potential of -84 mV (top). Percentage inhibition of the current amplitude measured at 40 ms was evaluated for 3.34 mM TEA, which is the IC 50 ). In this apical neurone, the reduction was 63 %, whereas in the basal one it was 58 %. The histograms represent mean values ± S.E.M. from 11-13 measurements. Pipette solution: KCl. Bath solution: Tyrode solution. Dendrite length range: apical neurones, 8-28 μm; basal neurones, 50-79 μm.

    Article Snippet: After centrifugation, the supernatant was replaced with regular Tyrode solution (mM: 140 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 , 10 Hepes, 10 glucose, 10 sodium pyruvate, pH adjusted to 7.4 with NaOH) supplemented with DNase (0.1 mg ml−1 ; type I, Roche Diagnostics, Milano, Italy) and incubated for 15 min with agitation.

    Techniques: Mass Spectrometry, Inhibition, Transferring

    Biophysical and pharmacological properties of voltage-gated Na + currents ( I Na ) in apical and basal neurones A , current-voltage relationships. I Na values for each membrane potential ( V m ) were averaged within each group (40 apical neurones, 37 basal neurones). I Na activated at approximately -50 mV and peaked at about -25 mV in both apical and basal neurones. Pipette solution: KCl. Bath solution: Tyrode solution. B , voltage dependence of the steady-state inactivation of I Na in apical (○) and basal (•) neurones. A standard two-pulse voltage protocol was used for this analysis. Prepulses 300 ms in duration and of variable amplitude (from -134 mV to -24 mV) were applied prior to the test pulse to -34 mV. Neurones were held at -84 mV between trials. The magnitude of the current elicited by the test pulse (-34 mV) was normalized to its maximal value and plotted against the prepulse potential (bottom). Each point represents the mean ± S.E.M. of 13-15 measurements. Data were fitted to a Boltzmann equation. For apical neurones, the half-maximal voltage ( V 0.5 ) was -78 mV and the slope ( k ) was 11.7 mV. For basal neurons, V 0.5 was -79 mV and k was 11.8 mV. Pipette solution: CsCl. Bath solution: Tyrode solution. Dendrite length range in these experiments: apical neurones, 12-35 μm; basal neurones, 52-96 μm. C , TTX sensitivity of I Na recorded in apical and basal neurones. Na + current was elicited by a test pulse to -34 mV after hyperpolarizing the membrane to -124 mV for 300 ms (top). Percentage inhibition of the maximal Na + current was evaluated for 15 nM TTX, which is the IC 50 ). The histograms represent mean values ± S.E.M. from 14 measurements. Pipette solution: CsCl. Bath solution: Tyrode solution. Dendrite length range: apical neurones, 14-35 μm; basal neurones, 49-102 μm.

    Journal: The Journal of Physiology

    Article Title: Apical and basal neurones isolated from the mouse vomeronasal organ differ for voltage-dependent currents

    doi: 10.1113/jphysiol.2003.052035

    Figure Lengend Snippet: Biophysical and pharmacological properties of voltage-gated Na + currents ( I Na ) in apical and basal neurones A , current-voltage relationships. I Na values for each membrane potential ( V m ) were averaged within each group (40 apical neurones, 37 basal neurones). I Na activated at approximately -50 mV and peaked at about -25 mV in both apical and basal neurones. Pipette solution: KCl. Bath solution: Tyrode solution. B , voltage dependence of the steady-state inactivation of I Na in apical (○) and basal (•) neurones. A standard two-pulse voltage protocol was used for this analysis. Prepulses 300 ms in duration and of variable amplitude (from -134 mV to -24 mV) were applied prior to the test pulse to -34 mV. Neurones were held at -84 mV between trials. The magnitude of the current elicited by the test pulse (-34 mV) was normalized to its maximal value and plotted against the prepulse potential (bottom). Each point represents the mean ± S.E.M. of 13-15 measurements. Data were fitted to a Boltzmann equation. For apical neurones, the half-maximal voltage ( V 0.5 ) was -78 mV and the slope ( k ) was 11.7 mV. For basal neurons, V 0.5 was -79 mV and k was 11.8 mV. Pipette solution: CsCl. Bath solution: Tyrode solution. Dendrite length range in these experiments: apical neurones, 12-35 μm; basal neurones, 52-96 μm. C , TTX sensitivity of I Na recorded in apical and basal neurones. Na + current was elicited by a test pulse to -34 mV after hyperpolarizing the membrane to -124 mV for 300 ms (top). Percentage inhibition of the maximal Na + current was evaluated for 15 nM TTX, which is the IC 50 ). The histograms represent mean values ± S.E.M. from 14 measurements. Pipette solution: CsCl. Bath solution: Tyrode solution. Dendrite length range: apical neurones, 14-35 μm; basal neurones, 49-102 μm.

    Article Snippet: After centrifugation, the supernatant was replaced with regular Tyrode solution (mM: 140 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 , 10 Hepes, 10 glucose, 10 sodium pyruvate, pH adjusted to 7.4 with NaOH) supplemented with DNase (0.1 mg ml−1 ; type I, Roche Diagnostics, Milano, Italy) and incubated for 15 min with agitation.

    Techniques: Transferring, Mass Spectrometry, Inhibition

    Amplitude distribution of voltage-gated Na + and K + currents ( I Na and I K , respectively) in apical and basal neurones A , distribution of peak values for I Na reveals that more than 70 % of apical neurones had I Na > 500 pA. In contrast, basal neurones typically had I Na of small amplitude. Note that the median (the horizontal line in the middle of each box) is larger in apical neurones than in basal neurones. Cells were held at -84 mV and depolarized, in 10 mV increments, to elicit maximal I Na . Pipette solution: KCl. Bath solution: Tyrode solution. B , amplitude distribution of I K . Current amplitude was evaluated at +46 mV and at the end of a 40 ms voltage step. Holding potential: -84 mV. Solutions as in A . Note that both the median and the data range are similar in the two neuronal subsets.

    Journal: The Journal of Physiology

    Article Title: Apical and basal neurones isolated from the mouse vomeronasal organ differ for voltage-dependent currents

    doi: 10.1113/jphysiol.2003.052035

    Figure Lengend Snippet: Amplitude distribution of voltage-gated Na + and K + currents ( I Na and I K , respectively) in apical and basal neurones A , distribution of peak values for I Na reveals that more than 70 % of apical neurones had I Na > 500 pA. In contrast, basal neurones typically had I Na of small amplitude. Note that the median (the horizontal line in the middle of each box) is larger in apical neurones than in basal neurones. Cells were held at -84 mV and depolarized, in 10 mV increments, to elicit maximal I Na . Pipette solution: KCl. Bath solution: Tyrode solution. B , amplitude distribution of I K . Current amplitude was evaluated at +46 mV and at the end of a 40 ms voltage step. Holding potential: -84 mV. Solutions as in A . Note that both the median and the data range are similar in the two neuronal subsets.

    Article Snippet: After centrifugation, the supernatant was replaced with regular Tyrode solution (mM: 140 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 , 10 Hepes, 10 glucose, 10 sodium pyruvate, pH adjusted to 7.4 with NaOH) supplemented with DNase (0.1 mg ml−1 ; type I, Roche Diagnostics, Milano, Italy) and incubated for 15 min with agitation.

    Techniques: Transferring, Mass Spectrometry

    Voltage-gated Na + and K + currents in apical and basal neurones A , differential interference contrast photomicrographs (left) of an apical neurone isolated from mouse VNO. Note the large dendritic knob. Dendrite length: 16 μm. Patch-clamp recording from this neurone (right) revealed the presence of TTX-sensitive, voltage-gated Na + currents (downward deflection in the current records) and of TEA-sensitive, voltage-gated K + currents (delayed, upward deflections in the current records). The cell was held at -84 mV and stepped in 10 mV increments from -74 mV to +46 mV. Pipette solution: KCl. Bath solution: Tyrode solution. B , a typical basal neurone with a long dendritic process (78 μm) expressing both TTX-sensitive, voltage-gated Na + currents and TEA-sensitive, voltage-gated K + currents. Voltage protocol and solutions as in A . Note that K + currents were of similar amplitude in both cells. In contrast, Na + currents were larger in apical neurones than in basal ones. Scale bar: 15 μm.

    Journal: The Journal of Physiology

    Article Title: Apical and basal neurones isolated from the mouse vomeronasal organ differ for voltage-dependent currents

    doi: 10.1113/jphysiol.2003.052035

    Figure Lengend Snippet: Voltage-gated Na + and K + currents in apical and basal neurones A , differential interference contrast photomicrographs (left) of an apical neurone isolated from mouse VNO. Note the large dendritic knob. Dendrite length: 16 μm. Patch-clamp recording from this neurone (right) revealed the presence of TTX-sensitive, voltage-gated Na + currents (downward deflection in the current records) and of TEA-sensitive, voltage-gated K + currents (delayed, upward deflections in the current records). The cell was held at -84 mV and stepped in 10 mV increments from -74 mV to +46 mV. Pipette solution: KCl. Bath solution: Tyrode solution. B , a typical basal neurone with a long dendritic process (78 μm) expressing both TTX-sensitive, voltage-gated Na + currents and TEA-sensitive, voltage-gated K + currents. Voltage protocol and solutions as in A . Note that K + currents were of similar amplitude in both cells. In contrast, Na + currents were larger in apical neurones than in basal ones. Scale bar: 15 μm.

    Article Snippet: After centrifugation, the supernatant was replaced with regular Tyrode solution (mM: 140 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 , 10 Hepes, 10 glucose, 10 sodium pyruvate, pH adjusted to 7.4 with NaOH) supplemented with DNase (0.1 mg ml−1 ; type I, Roche Diagnostics, Milano, Italy) and incubated for 15 min with agitation.

    Techniques: Isolation, Patch Clamp, Transferring, Expressing

    PP2A is involved in VT-1-induced apoptosis. (A) Ramos cells were incubated with okadaic acid (50 nM) or with vehicle (DMSO) or without any treatment (−) for 1 h before being treated with VT-1 for 4 h. The cells were labeled with annexin V-FITC and PI and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. (B) VT-1-sensitive or -resistant cells were incubated with or without VT-1 (5 ng/ml) for various periods of time and lysed. The lysates were immunoprecipitated with an anti-PP2A Ab, and the levels of PP2A activity were determined by measuring the release of phosphate from a phosphopeptide substrate in a colorimetric assay. The increases in the PP2A activity levels of treated samples were determined with respect to the levels in untreated samples. Error bars indicate the standard deviations.

    Journal: Journal of Virology

    Article Title: Truncated Form of the Epstein-Barr Virus Protein EBNA-LP Protects against Caspase-Dependent Apoptosis by Inhibiting Protein Phosphatase 2A ▿

    doi: 10.1128/JVI.02435-06

    Figure Lengend Snippet: PP2A is involved in VT-1-induced apoptosis. (A) Ramos cells were incubated with okadaic acid (50 nM) or with vehicle (DMSO) or without any treatment (−) for 1 h before being treated with VT-1 for 4 h. The cells were labeled with annexin V-FITC and PI and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. (B) VT-1-sensitive or -resistant cells were incubated with or without VT-1 (5 ng/ml) for various periods of time and lysed. The lysates were immunoprecipitated with an anti-PP2A Ab, and the levels of PP2A activity were determined by measuring the release of phosphate from a phosphopeptide substrate in a colorimetric assay. The increases in the PP2A activity levels of treated samples were determined with respect to the levels in untreated samples. Error bars indicate the standard deviations.

    Article Snippet: The cells were washed in phosphate-buffered saline (PBS), resuspended in annexin buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 ) containing 2.5 μg/ml fluorescein isothiocyanate (FITC)-labeled annexin V (Roche Applied Science), and incubated at 4°C for 5 to 10 min.

    Techniques: Incubation, Labeling, Flow Cytometry, Cytometry, Immunoprecipitation, Activity Assay, Colorimetric Assay

    Effect of stable transfection of EBNA-LP on apoptosis. Ramos cells were cotransfected with EBNA-LP expression vectors and the pSG5(Neo R ) vector, which carries the neomycin resistance gene. The cells were grown in selection medium (complete RPMI supplemented with 16 mg/ml neomycin) for 4 weeks, and the clones were then tested. (A) Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb. Molecular sizes are indicated to the right. (B) Stable transfectants were treated with VT-1 (5 ng/ml) for 16 h, labeled with annexin V-FITC and PI, and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. The percentages of apoptosis inhibition were determined by comparison with cells transfected with the pSG5 vector. Error bars shown the standard deviations.

    Journal: Journal of Virology

    Article Title: Truncated Form of the Epstein-Barr Virus Protein EBNA-LP Protects against Caspase-Dependent Apoptosis by Inhibiting Protein Phosphatase 2A ▿

    doi: 10.1128/JVI.02435-06

    Figure Lengend Snippet: Effect of stable transfection of EBNA-LP on apoptosis. Ramos cells were cotransfected with EBNA-LP expression vectors and the pSG5(Neo R ) vector, which carries the neomycin resistance gene. The cells were grown in selection medium (complete RPMI supplemented with 16 mg/ml neomycin) for 4 weeks, and the clones were then tested. (A) Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with 4D3 anti-EBNA-LP MAb. Molecular sizes are indicated to the right. (B) Stable transfectants were treated with VT-1 (5 ng/ml) for 16 h, labeled with annexin V-FITC and PI, and analyzed by using a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. The percentages of apoptosis inhibition were determined by comparison with cells transfected with the pSG5 vector. Error bars shown the standard deviations.

    Article Snippet: The cells were washed in phosphate-buffered saline (PBS), resuspended in annexin buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 ) containing 2.5 μg/ml fluorescein isothiocyanate (FITC)-labeled annexin V (Roche Applied Science), and incubated at 4°C for 5 to 10 min.

    Techniques: Stable Transfection, Expressing, Plasmid Preparation, Selection, Clone Assay, Electrophoresis, Labeling, Flow Cytometry, Cytometry, Inhibition, Transfection

    Infection with the P3HR1 strain of EBV protects BL cells against VT-1- and staurosporine-induced apoptosis. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.

    Journal: Journal of Virology

    Article Title: Truncated Form of the Epstein-Barr Virus Protein EBNA-LP Protects against Caspase-Dependent Apoptosis by Inhibiting Protein Phosphatase 2A ▿

    doi: 10.1128/JVI.02435-06

    Figure Lengend Snippet: Infection with the P3HR1 strain of EBV protects BL cells against VT-1- and staurosporine-induced apoptosis. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed, and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.

    Article Snippet: The cells were washed in phosphate-buffered saline (PBS), resuspended in annexin buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 ) containing 2.5 μg/ml fluorescein isothiocyanate (FITC)-labeled annexin V (Roche Applied Science), and incubated at 4°C for 5 to 10 min.

    Techniques: Infection, Incubation, Labeling, Flow Cytometry, Cytometry, Electrophoresis

    VT-1 and staurosporine induce caspase-dependent apoptotic cell death in some, but not all, BL cell lines. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.

    Journal: Journal of Virology

    Article Title: Truncated Form of the Epstein-Barr Virus Protein EBNA-LP Protects against Caspase-Dependent Apoptosis by Inhibiting Protein Phosphatase 2A ▿

    doi: 10.1128/JVI.02435-06

    Figure Lengend Snippet: VT-1 and staurosporine induce caspase-dependent apoptotic cell death in some, but not all, BL cell lines. (A) Cells were incubated for 16 h with VT-1 (5 ng/ml), staurosporine (2.5 μM), or complete RPMI medium (control). The cells were labeled with annexin V-FITC and PI and analyzed with a FACSCalibur flow cytometer to determine the percentages of apoptotic cells. Error bars show the standard deviations. (B) Cells were incubated for 16 h with VT-1, staurosporine, or complete RPMI medium (−). Cell pellets were lysed and equal amounts of protein were subjected to electrophoresis in 12% bis-Tris precast gels. The proteins were transferred to PVDF membranes, which were probed with an anti-caspase 8 MAb or an anti-PARP MAb. Molecular sizes are indicated to the right of each gel.

    Article Snippet: The cells were washed in phosphate-buffered saline (PBS), resuspended in annexin buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 ) containing 2.5 μg/ml fluorescein isothiocyanate (FITC)-labeled annexin V (Roche Applied Science), and incubated at 4°C for 5 to 10 min.

    Techniques: Incubation, Labeling, Flow Cytometry, Cytometry, Electrophoresis

    Quantifying changes in DNase I-sensitivity with qrt-PCR. qrt-PCR was used to detected small changes in DNase I-sensitivity in two reciprocally expressed genes ( redD and rlpA ) at the earliest and latest time points. In vivo digestions were performed as described in Figure 1 ; qrt-PCR is described elsewhere ( 6 ). DNase I sensitivity was estimated by the percentage loss of copy number after the rate of digestion had reached a steady level (commonly after 4 U of enzyme had been used).

    Journal: Nucleic Acids Research

    Article Title: In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

    doi: 10.1093/nar/gkl649

    Figure Lengend Snippet: Quantifying changes in DNase I-sensitivity with qrt-PCR. qrt-PCR was used to detected small changes in DNase I-sensitivity in two reciprocally expressed genes ( redD and rlpA ) at the earliest and latest time points. In vivo digestions were performed as described in Figure 1 ; qrt-PCR is described elsewhere ( 6 ). DNase I sensitivity was estimated by the percentage loss of copy number after the rate of digestion had reached a steady level (commonly after 4 U of enzyme had been used).

    Article Snippet: The cells were washed in the same volume of DNase I Digestion Buffer [DDB: 10 mM Tris–HCl, 2.6% sucrose, 10 mM MgCl2 , 0.25 mM CaCl2 , 0.1 mM DTT, 0.5% NP-40 and 0.5% Triton X-100), and split into five aliquots and digested with different amounts of DNase I (0 to 50 U of Roche's molecular biology grade enzyme) at 37°C for 3 min.

    Techniques: Quantitative RT-PCR, In Vivo

    Transcriptionally regulated genes in S.coelicolor show a positive correlation between DNase I-sensitivity and level of expression. qrt-PCR was used to measure both relative DNase I sensitivity and RNA abundance ( Figure 1 , lower) for each of the twelve genes used in this study at the three timepoints described. Expression data is relative to the value of act II-orf4 gene at T3 timepoint. Each point is the average of at least three independent measurements with standard deviations shown as dotted lines.

    Journal: Nucleic Acids Research

    Article Title: In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

    doi: 10.1093/nar/gkl649

    Figure Lengend Snippet: Transcriptionally regulated genes in S.coelicolor show a positive correlation between DNase I-sensitivity and level of expression. qrt-PCR was used to measure both relative DNase I sensitivity and RNA abundance ( Figure 1 , lower) for each of the twelve genes used in this study at the three timepoints described. Expression data is relative to the value of act II-orf4 gene at T3 timepoint. Each point is the average of at least three independent measurements with standard deviations shown as dotted lines.

    Article Snippet: The cells were washed in the same volume of DNase I Digestion Buffer [DDB: 10 mM Tris–HCl, 2.6% sucrose, 10 mM MgCl2 , 0.25 mM CaCl2 , 0.1 mM DTT, 0.5% NP-40 and 0.5% Triton X-100), and split into five aliquots and digested with different amounts of DNase I (0 to 50 U of Roche's molecular biology grade enzyme) at 37°C for 3 min.

    Techniques: Expressing, Quantitative RT-PCR, Activated Clotting Time Assay

    In vivo DNase I digestion of the S.coelicolor genome results in fractionation according to transcriptional activity. ( A ) HCHO-crosslinked DNA–protein complexes from S.coelicolor collected at T1 (shown as an example with the triangle representing increasing amounts of DNase I) and T3 were digested in vivo with increasing amounts of DNase I, and the nucleoprotein complexes separated by 0.7% agarose gel electrophoresis. DNA was extracted from the soluble fraction, defined as that resolved by the gel and indicated by a box, and used as a probe in the subsequent slot-blot experiment. ( B ) DNA and RNA samples were labeled with DIG-dUTP and used as probes in slot-blot hybridizations with 12 ∼500 bp PCR products corresponding to the panel of genes used in the experiment. At each time point, the RNA and DNA probes detect the same targets, demonstrating that in vivo DNase I treatment had fractionated the genome based on transcriptional activity. Different targets were detected by samples from time points T1 and T3, consistent with the transcriptional programme changing as S.coelicolor enters stationary phase.

    Journal: Nucleic Acids Research

    Article Title: In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

    doi: 10.1093/nar/gkl649

    Figure Lengend Snippet: In vivo DNase I digestion of the S.coelicolor genome results in fractionation according to transcriptional activity. ( A ) HCHO-crosslinked DNA–protein complexes from S.coelicolor collected at T1 (shown as an example with the triangle representing increasing amounts of DNase I) and T3 were digested in vivo with increasing amounts of DNase I, and the nucleoprotein complexes separated by 0.7% agarose gel electrophoresis. DNA was extracted from the soluble fraction, defined as that resolved by the gel and indicated by a box, and used as a probe in the subsequent slot-blot experiment. ( B ) DNA and RNA samples were labeled with DIG-dUTP and used as probes in slot-blot hybridizations with 12 ∼500 bp PCR products corresponding to the panel of genes used in the experiment. At each time point, the RNA and DNA probes detect the same targets, demonstrating that in vivo DNase I treatment had fractionated the genome based on transcriptional activity. Different targets were detected by samples from time points T1 and T3, consistent with the transcriptional programme changing as S.coelicolor enters stationary phase.

    Article Snippet: The cells were washed in the same volume of DNase I Digestion Buffer [DDB: 10 mM Tris–HCl, 2.6% sucrose, 10 mM MgCl2 , 0.25 mM CaCl2 , 0.1 mM DTT, 0.5% NP-40 and 0.5% Triton X-100), and split into five aliquots and digested with different amounts of DNase I (0 to 50 U of Roche's molecular biology grade enzyme) at 37°C for 3 min.

    Techniques: In Vivo, Fractionation, Activity Assay, Agarose Gel Electrophoresis, Dot Blot, Labeling, Polymerase Chain Reaction

    Gene expression during growth of  S.coelicolor  strain M145. Spores were inoculated into R5 liquid medium and grown at 30°C for 3 days. The culture was monitored throughout the experiment for growth and production of the two pigmented antibiotics Act and Red (5; upper). Mycelium was harvested at the three time points indicated [early growth phase (T1), transition phase (T2) and stationary phase (T3)] to generate RNA samples for expression analysis by qrt-PCR (lower) and for use in subsequent DNase I-sensitivity experiments.

    Journal: Nucleic Acids Research

    Article Title: In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

    doi: 10.1093/nar/gkl649

    Figure Lengend Snippet: Gene expression during growth of S.coelicolor strain M145. Spores were inoculated into R5 liquid medium and grown at 30°C for 3 days. The culture was monitored throughout the experiment for growth and production of the two pigmented antibiotics Act and Red (5; upper). Mycelium was harvested at the three time points indicated [early growth phase (T1), transition phase (T2) and stationary phase (T3)] to generate RNA samples for expression analysis by qrt-PCR (lower) and for use in subsequent DNase I-sensitivity experiments.

    Article Snippet: The cells were washed in the same volume of DNase I Digestion Buffer [DDB: 10 mM Tris–HCl, 2.6% sucrose, 10 mM MgCl2 , 0.25 mM CaCl2 , 0.1 mM DTT, 0.5% NP-40 and 0.5% Triton X-100), and split into five aliquots and digested with different amounts of DNase I (0 to 50 U of Roche's molecular biology grade enzyme) at 37°C for 3 min.

    Techniques: Expressing, Activated Clotting Time Assay, Quantitative RT-PCR

    In vivo DNase I sensitivity determined by Southern hybridization analysis. DNA harvested from mycelium taken from each time point was treated with increasing amounts of DNase I, digested to completion with restriction enzymes and used in Southern hybridization assays. Blots were hybridized with mixtures of probes for ( A ) rbnH , redD , rlpA and sti1 and ( B ) act II-orf4, afsQ2 , hrdB and rpmG3 . The positions of the genomic bands for each gene are indicated, with the amount of DNase I used increasing from left to right.

    Journal: Nucleic Acids Research

    Article Title: In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure

    doi: 10.1093/nar/gkl649

    Figure Lengend Snippet: In vivo DNase I sensitivity determined by Southern hybridization analysis. DNA harvested from mycelium taken from each time point was treated with increasing amounts of DNase I, digested to completion with restriction enzymes and used in Southern hybridization assays. Blots were hybridized with mixtures of probes for ( A ) rbnH , redD , rlpA and sti1 and ( B ) act II-orf4, afsQ2 , hrdB and rpmG3 . The positions of the genomic bands for each gene are indicated, with the amount of DNase I used increasing from left to right.

    Article Snippet: The cells were washed in the same volume of DNase I Digestion Buffer [DDB: 10 mM Tris–HCl, 2.6% sucrose, 10 mM MgCl2 , 0.25 mM CaCl2 , 0.1 mM DTT, 0.5% NP-40 and 0.5% Triton X-100), and split into five aliquots and digested with different amounts of DNase I (0 to 50 U of Roche's molecular biology grade enzyme) at 37°C for 3 min.

    Techniques: In Vivo, Hybridization, Activated Clotting Time Assay