lysis buffer  (Thermo Fisher)


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    Structured Review

    Thermo Fisher lysis buffer
    Lysis Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5963 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lysis buffer/product/Thermo Fisher
    Average 99 stars, based on 5963 article reviews
    Price from $9.99 to $1999.99
    lysis buffer - by Bioz Stars, 2020-03
    99/100 stars

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    Related Articles

    Centrifugation:

    Article Title: EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming
    Article Snippet: .. Co-immunoprecipitation Cellular lysates were prepared by incubating the cells in lysis buffer (10 mM CHAPS, 50 mM Tris-HCL, pH 8, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) containing a protease inhibitor cocktail (Thermo Fisher Scientific) for 20 min at 4 °C, followed by centrifugation at 14,000g for 15 min at 4 °C. .. The protein concentration of the lysates was determined using the Bradford protein assay kit (Bio-Rad) according to the manufacturer’s protocol.

    Article Title: Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD
    Article Snippet: Cells were scraped into ice-cold PBS and collected by centrifugation. .. Cell pellets were suspended in 200 μl of lysis buffer containing Protease Inhibitor Mixture (Thermo Scientific).

    Article Title: Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking
    Article Snippet: .. In brief, the cells were collected by centrifugation and resuspended in lysis buffer consisting of 200 mM NaCl, 10 mM Tris–HCl pH 7.4, 2 mM EDTA pH 8.0, 0.2% (w/v), sodium dodecyl sulphate (SDS) and freshly added proteinase K (Thermo Fisher Scientific; Cat. No.: #EO0491) at a final concentration of 200 ng ml−1 . .. After an overnight incubation period at 56°C, the DNA was precipitated by adding isopropanol (1:1) and immediate mixing of the aqueous and organic phases.

    Article Title: MicroRNA-29 family functions as a tumor suppressor by targeting RPS15A and regulating cell cycle in hepatocellular carcinoma
    Article Snippet: The cytoplasmic lysate was isolated by centrifugation at 10,000× for 15 min and supernatant was collected. .. Protein kinase K (20 mg/mL) and 1 μL of 10% SDS in 100 μL of lysis buffer were added to the pellet and incubated at 55°C for 20 min. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated with Trizol reagent (Invitrogen).

    Amplification:

    Article Title: Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis
    Article Snippet: .. Cell sorting, reverse transcription, amplification, and sequencing One thousand cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL−1 RNase Inhibitor (Takara). .. Transcriptome amplifications were performed according to Smart-Seq2 protocol [ ] with modification of reagent amount and PCR cycle numbers.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression
    Article Snippet: Cells were lysed in 100μl of lysis buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA and 1% NP40), and protein concentration in cell lysate was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific). .. A total of 30ug of proteins were separated in SDS-12% polyacrylamide gel (PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane.

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
    Article Snippet: The beads were washed three times with lysis buffer, and the precipitates were eluted in 30 μl of M-PER mammalian protein extraction reagent (Thermo, Rockford, IL) and 6× loading buffer by boiling at 95°C for 5 min. .. The beads were spun down in a microcentrifuge at full speed at 4°C for 5 min, and the supernatants were subjected to SDS-10% PAGE.

    Blocking Assay:

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
    Article Snippet: The beads were washed three times with lysis buffer, and the precipitates were eluted in 30 μl of M-PER mammalian protein extraction reagent (Thermo, Rockford, IL) and 6× loading buffer by boiling at 95°C for 5 min. .. The resolved proteins were transferred to an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore), and the membranes were incubated with Tris-buffered saline with Tween 20 (TBST) blocking buffer (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20 containing 5% skim milk powder) for 1 h at room temperature, followed by further incubation with primary antibody at 4°C overnight.

    Electrophoresis:

    Article Title: The effect of NAMPT deletion in projection neurons on the function and structure of neuromuscular junction (NMJ) in mice
    Article Snippet: Briefly, the fresh muscle tissues were dissolved in lysis buffer mixed with protease inhibitor (Cat. No. 36978, Pierce Biotechnology, Rockford, IL) and cocktails of phosphatase inhibitor (Cat. No. P8340 Sigma-Aldrich), and homogenized. .. The protein samples were boiled for 5 minutes, loaded in 10% SDS-polyacrylamide gels, subjected to electrophoresis at 100 mV for 100–110 minutes, and transferred to polyvinylidene fluoride membranes using Mixed Molecular Weight function of Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA).

    Article Title: Membralin deficiency dysregulates astrocytic glutamate homeostasis, leading to ALS-like impairment
    Article Snippet: Spinal cord or astrocyte samples were homogenized in cold lysis buffer comprising 1% NP40, 1% digitonin, 50 mM Tris-HCl (pH 8.0), and 150 mM NaCl supplemented with Halt Protease and Phosphatase Inhibitor Cocktails (Thermo Fisher Scientific). .. Protein (10–25 μg) was subjected to electrophoresis using 4%–20% TGX precast protein gels (BioRad), transferred to PVDF membranes, and probed with the indicated primary antibodies.

    Incubation:

    Article Title: EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming
    Article Snippet: Co-immunoprecipitation Cellular lysates were prepared by incubating the cells in lysis buffer (10 mM CHAPS, 50 mM Tris-HCL, pH 8, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) containing a protease inhibitor cocktail (Thermo Fisher Scientific) for 20 min at 4 °C, followed by centrifugation at 14,000g for 15 min at 4 °C. .. For immunoprecipitation, 500 µg of protein was incubated with 2 µg specific antibodies for 12 h at 4 °C with constant rotation; 60 µl of 50% protein A or G agarose beads was then added and the incubation was continued for an additional 2 h. Beads were then washed five times using CHAPS co-IP lysis buffer.

    Article Title: Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression
    Article Snippet: Cells were lysed in 100μl of lysis buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA and 1% NP40), and protein concentration in cell lysate was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific). .. The blot was incubated at 4°C overnight with polyclonal rabbit anti-HBc (Dako, 1:3000), anti-HBs (Novus, 1:3000) diluted in 5% milk/TBST.

    Article Title: Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD
    Article Snippet: After two washes with ice-cold PBS, the cells were incubated in 8 ml of ice-cold PBS with 400 μl of quenching solution for 10 min at 4 °C and washed again with ice-cold PBS. .. Cell pellets were suspended in 200 μl of lysis buffer containing Protease Inhibitor Mixture (Thermo Scientific).

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
    Article Snippet: Thirty microliters of protein G agarose beads (EMD Millipore, Temecula, CA) was added, and the mixture was further incubated for at least 2 h at 4°C. .. The beads were washed three times with lysis buffer, and the precipitates were eluted in 30 μl of M-PER mammalian protein extraction reagent (Thermo, Rockford, IL) and 6× loading buffer by boiling at 95°C for 5 min.

    Article Title: The effect of NAMPT deletion in projection neurons on the function and structure of neuromuscular junction (NMJ) in mice
    Article Snippet: Briefly, the fresh muscle tissues were dissolved in lysis buffer mixed with protease inhibitor (Cat. No. 36978, Pierce Biotechnology, Rockford, IL) and cocktails of phosphatase inhibitor (Cat. No. P8340 Sigma-Aldrich), and homogenized. .. The membranes were blocked for 1 hour by 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBS-T), and were then incubated overnight at 4 °C with mouse monoclonal anti-NAMPT (1:1000, ALX-804-922-C100, Enzo Life Sciences, Farmingdale, NY) in 1% (w/v) BSA.

    Article Title: Sensory Experience Remodels Genome Architecture in Neural Circuit to Drive Motor Learning
    Article Snippet: .. For chromatin or nucleocytoplasmic fractionation, tissues were homogenized in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton-X, RNaseOUT (Thermo Fisher Scientific)) using a syringe and incubated on ice for 10 minutes. .. After spinning down, the RNA in the supernatant (nucleocytoplasmic fraction) was extracted with Trizol LS (Thermo Fisher Scientific).

    Article Title: Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking
    Article Snippet: In brief, the cells were collected by centrifugation and resuspended in lysis buffer consisting of 200 mM NaCl, 10 mM Tris–HCl pH 7.4, 2 mM EDTA pH 8.0, 0.2% (w/v), sodium dodecyl sulphate (SDS) and freshly added proteinase K (Thermo Fisher Scientific; Cat. No.: #EO0491) at a final concentration of 200 ng ml−1 . .. After an overnight incubation period at 56°C, the DNA was precipitated by adding isopropanol (1:1) and immediate mixing of the aqueous and organic phases.

    Article Title: MicroRNA-29 family functions as a tumor suppressor by targeting RPS15A and regulating cell cycle in hepatocellular carcinoma
    Article Snippet: .. Protein kinase K (20 mg/mL) and 1 μL of 10% SDS in 100 μL of lysis buffer were added to the pellet and incubated at 55°C for 20 min. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated with Trizol reagent (Invitrogen). .. The levels of RPS15A in the Bio-miR-29a, b, c pull-down were quantified by qRT-PCR.

    Expressing:

    Article Title: Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression
    Article Snippet: Paragraph title: 2.2. Transient transfection and detection of protein expression ... Cells were lysed in 100μl of lysis buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA and 1% NP40), and protein concentration in cell lysate was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific).

    Article Title: Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling
    Article Snippet: .. For cellular protein expression determination, cells were scraped into ice-cold lysis buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and protease and phosphatase inhibitors (Halt 78447; Thermo Fisher Scientific). .. The lysates were then triturated through a small-bore needle using a syringe, sonicated, and kept on ice.

    BIA-KA:

    Article Title: Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression
    Article Snippet: .. Cells were lysed in 100μl of lysis buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA and 1% NP40), and protein concentration in cell lysate was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific). .. Core and envelope proteins in cell lysate were detected by Western blot analysis.

    Article Title: The effect of NAMPT deletion in projection neurons on the function and structure of neuromuscular junction (NMJ) in mice
    Article Snippet: Briefly, the fresh muscle tissues were dissolved in lysis buffer mixed with protease inhibitor (Cat. No. 36978, Pierce Biotechnology, Rockford, IL) and cocktails of phosphatase inhibitor (Cat. No. P8340 Sigma-Aldrich), and homogenized. .. The protein concentration of the cell lysate was measured by bicinchoninic acid (BCA) protein assay kit (Cat. No. 23277, Pierce Biotechnology).

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma). .. Protein concentration was measured using the BCA Protein Assay kit (Pierce).

    Article Title: Membralin deficiency dysregulates astrocytic glutamate homeostasis, leading to ALS-like impairment
    Article Snippet: Spinal cord or astrocyte samples were homogenized in cold lysis buffer comprising 1% NP40, 1% digitonin, 50 mM Tris-HCl (pH 8.0), and 150 mM NaCl supplemented with Halt Protease and Phosphatase Inhibitor Cocktails (Thermo Fisher Scientific). .. Protein concentrations were determined by bicinchoninic (BCA) assay, whereby protein samples were subjected to immunoblot analysis.

    Modification:

    Article Title: Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression
    Article Snippet: Huh7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum. .. Cells were lysed in 100μl of lysis buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA and 1% NP40), and protein concentration in cell lysate was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific).

    Article Title: Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis
    Article Snippet: Cell sorting, reverse transcription, amplification, and sequencing One thousand cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL−1 RNase Inhibitor (Takara). .. Transcriptome amplifications were performed according to Smart-Seq2 protocol [ ] with modification of reagent amount and PCR cycle numbers.

    Western Blot:

    Article Title: Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression
    Article Snippet: Cells were lysed in 100μl of lysis buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA and 1% NP40), and protein concentration in cell lysate was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific). .. Core and envelope proteins in cell lysate were detected by Western blot analysis.

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
    Article Snippet: Paragraph title: Coimmunoprecipitation and Western blotting. ... The beads were washed three times with lysis buffer, and the precipitates were eluted in 30 μl of M-PER mammalian protein extraction reagent (Thermo, Rockford, IL) and 6× loading buffer by boiling at 95°C for 5 min.

    Article Title: The effect of NAMPT deletion in projection neurons on the function and structure of neuromuscular junction (NMJ) in mice
    Article Snippet: Paragraph title: Western blotting analysis ... Briefly, the fresh muscle tissues were dissolved in lysis buffer mixed with protease inhibitor (Cat. No. 36978, Pierce Biotechnology, Rockford, IL) and cocktails of phosphatase inhibitor (Cat. No. P8340 Sigma-Aldrich), and homogenized.

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Paragraph title: Transfection, Immunoprecipitation, Co-immunoprecipitation and Western Blot ... Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma).

    Transfection:

    Article Title: Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression
    Article Snippet: Paragraph title: 2.2. Transient transfection and detection of protein expression ... Cells were lysed in 100μl of lysis buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA and 1% NP40), and protein concentration in cell lysate was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific).

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Paragraph title: Transfection, Immunoprecipitation, Co-immunoprecipitation and Western Blot ... Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma).

    Article Title: Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking
    Article Snippet: Orthogonal HTGTS sample preparation Transfections for generating genomic DNA samples for orthogonal HTGTS analyses were carried out in HEK293T cells and iPSCs ( , respectively). .. In brief, the cells were collected by centrifugation and resuspended in lysis buffer consisting of 200 mM NaCl, 10 mM Tris–HCl pH 7.4, 2 mM EDTA pH 8.0, 0.2% (w/v), sodium dodecyl sulphate (SDS) and freshly added proteinase K (Thermo Fisher Scientific; Cat. No.: #EO0491) at a final concentration of 200 ng ml−1 .

    Immunoprecipitation:

    Article Title: EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming
    Article Snippet: Co-immunoprecipitation Cellular lysates were prepared by incubating the cells in lysis buffer (10 mM CHAPS, 50 mM Tris-HCL, pH 8, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) containing a protease inhibitor cocktail (Thermo Fisher Scientific) for 20 min at 4 °C, followed by centrifugation at 14,000g for 15 min at 4 °C. .. For immunoprecipitation, 500 µg of protein was incubated with 2 µg specific antibodies for 12 h at 4 °C with constant rotation; 60 µl of 50% protein A or G agarose beads was then added and the incubation was continued for an additional 2 h. Beads were then washed five times using CHAPS co-IP lysis buffer.

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Paragraph title: Transfection, Immunoprecipitation, Co-immunoprecipitation and Western Blot ... Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma).

    Protease Inhibitor:

    Article Title: EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming
    Article Snippet: .. Co-immunoprecipitation Cellular lysates were prepared by incubating the cells in lysis buffer (10 mM CHAPS, 50 mM Tris-HCL, pH 8, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) containing a protease inhibitor cocktail (Thermo Fisher Scientific) for 20 min at 4 °C, followed by centrifugation at 14,000g for 15 min at 4 °C. .. The protein concentration of the lysates was determined using the Bradford protein assay kit (Bio-Rad) according to the manufacturer’s protocol.

    Article Title: Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD
    Article Snippet: .. Cell pellets were suspended in 200 μl of lysis buffer containing Protease Inhibitor Mixture (Thermo Scientific). .. Cell lysates were collected after centrifugation at 10,000 × g for 2 min at 4 °C.

    Article Title: Excised linear introns regulate growth in yeast
    Article Snippet: .. Crude lysates were prepared by re-suspending an aliquot of thawed lysate powder (500–800 μL of loosely packed powder) in 1 mL of lysis buffer (10 mM Tris-HCl [pH 7.4], 5 mM MgCl2 , 100 mM KCl, 1% Triton X−100, 1% Sodium Deoxycholate, 2 mM DTT, 20 U/ml SUPERase•In [Ambion], cOmplete EDTA-free Protease Inhibitor Cocktail [Roche]). ..

    Article Title: The effect of NAMPT deletion in projection neurons on the function and structure of neuromuscular junction (NMJ) in mice
    Article Snippet: .. Briefly, the fresh muscle tissues were dissolved in lysis buffer mixed with protease inhibitor (Cat. No. 36978, Pierce Biotechnology, Rockford, IL) and cocktails of phosphatase inhibitor (Cat. No. P8340 Sigma-Aldrich), and homogenized. .. The protein concentration of the cell lysate was measured by bicinchoninic acid (BCA) protein assay kit (Cat. No. 23277, Pierce Biotechnology).

    Dissection:

    Article Title: Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling
    Article Snippet: For cellular protein expression determination, cells were scraped into ice-cold lysis buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and protease and phosphatase inhibitors (Halt 78447; Thermo Fisher Scientific). .. For pulmonary tissue protein expression analysis, mouse pups were euthanized using 200 mg/kg pentobarbital sodium intraperitoneal injection, and whole lung tissues were obtained by dissection and frozen in liquid N2 .

    Cell Culture:

    Article Title: Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression
    Article Snippet: Huh7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum. .. Cells were lysed in 100μl of lysis buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA and 1% NP40), and protein concentration in cell lysate was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific).

    Article Title: Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD
    Article Snippet: Cells cultured in 100 mm dishes were washed twice with 8 ml of ice-cold PBS, and then incubated with 8 ml of ice-cold PBS containing sulfo-NHS-SS-Biotin for 30 min at 4 °C. .. Cell pellets were suspended in 200 μl of lysis buffer containing Protease Inhibitor Mixture (Thermo Scientific).

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Seventy percent confluent HEK293 cell monolayers cultured on a 10 cm2 dishes were transfected with the RIPOR2-HA construct using Lipofectamine 3000 (Invitrogen) following the manufacturer’s standard instructions. .. Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma).

    Sedimentation:

    Article Title: Excised linear introns regulate growth in yeast
    Article Snippet: Paragraph title: Sedimentation velocity ... Crude lysates were prepared by re-suspending an aliquot of thawed lysate powder (500–800 μL of loosely packed powder) in 1 mL of lysis buffer (10 mM Tris-HCl [pH 7.4], 5 mM MgCl2 , 100 mM KCl, 1% Triton X−100, 1% Sodium Deoxycholate, 2 mM DTT, 20 U/ml SUPERase•In [Ambion], cOmplete EDTA-free Protease Inhibitor Cocktail [Roche]).

    Protein Concentration:

    Article Title: EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming
    Article Snippet: Co-immunoprecipitation Cellular lysates were prepared by incubating the cells in lysis buffer (10 mM CHAPS, 50 mM Tris-HCL, pH 8, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) containing a protease inhibitor cocktail (Thermo Fisher Scientific) for 20 min at 4 °C, followed by centrifugation at 14,000g for 15 min at 4 °C. .. The protein concentration of the lysates was determined using the Bradford protein assay kit (Bio-Rad) according to the manufacturer’s protocol.

    Article Title: Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression
    Article Snippet: .. Cells were lysed in 100μl of lysis buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA and 1% NP40), and protein concentration in cell lysate was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific). .. Core and envelope proteins in cell lysate were detected by Western blot analysis.

    Article Title: The effect of NAMPT deletion in projection neurons on the function and structure of neuromuscular junction (NMJ) in mice
    Article Snippet: Briefly, the fresh muscle tissues were dissolved in lysis buffer mixed with protease inhibitor (Cat. No. 36978, Pierce Biotechnology, Rockford, IL) and cocktails of phosphatase inhibitor (Cat. No. P8340 Sigma-Aldrich), and homogenized. .. The protein concentration of the cell lysate was measured by bicinchoninic acid (BCA) protein assay kit (Cat. No. 23277, Pierce Biotechnology).

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma). .. Protein concentration was measured using the BCA Protein Assay kit (Pierce).

    Sequencing:

    Article Title: Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis
    Article Snippet: .. Cell sorting, reverse transcription, amplification, and sequencing One thousand cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL−1 RNase Inhibitor (Takara). .. Transcriptome amplifications were performed according to Smart-Seq2 protocol [ ] with modification of reagent amount and PCR cycle numbers.

    Sonication:

    Article Title: Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling
    Article Snippet: For cellular protein expression determination, cells were scraped into ice-cold lysis buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and protease and phosphatase inhibitors (Halt 78447; Thermo Fisher Scientific). .. The lysates were then triturated through a small-bore needle using a syringe, sonicated, and kept on ice.

    Injection:

    Article Title: Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling
    Article Snippet: For cellular protein expression determination, cells were scraped into ice-cold lysis buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and protease and phosphatase inhibitors (Halt 78447; Thermo Fisher Scientific). .. For pulmonary tissue protein expression analysis, mouse pups were euthanized using 200 mg/kg pentobarbital sodium intraperitoneal injection, and whole lung tissues were obtained by dissection and frozen in liquid N2 .

    Transmission Electron Microscopy:

    Article Title: Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis
    Article Snippet: .. Cell sorting, reverse transcription, amplification, and sequencing One thousand cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL−1 RNase Inhibitor (Takara). .. Transcriptome amplifications were performed according to Smart-Seq2 protocol [ ] with modification of reagent amount and PCR cycle numbers.

    RNA Sequencing Assay:

    Article Title: Sensory Experience Remodels Genome Architecture in Neural Circuit to Drive Motor Learning
    Article Snippet: Paragraph title: RNA-Seq ... For chromatin or nucleocytoplasmic fractionation, tissues were homogenized in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton-X, RNaseOUT (Thermo Fisher Scientific)) using a syringe and incubated on ice for 10 minutes.

    Pull Down Assay:

    Article Title: MicroRNA-29 family functions as a tumor suppressor by targeting RPS15A and regulating cell cycle in hepatocellular carcinoma
    Article Snippet: Paragraph title: Biotin-labelled RNA pull-down assay ... Protein kinase K (20 mg/mL) and 1 μL of 10% SDS in 100 μL of lysis buffer were added to the pellet and incubated at 55°C for 20 min. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated with Trizol reagent (Invitrogen).

    Magnetic Beads:

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma). .. Immunoprecipitations were performed with anti-HA and anti-c-Myc coupled magnetic beads (Pierce) to avoid having immunoglobulins in the protein eluates.

    Article Title: MicroRNA-29 family functions as a tumor suppressor by targeting RPS15A and regulating cell cycle in hepatocellular carcinoma
    Article Snippet: The lysate was added to the strepatavidin-coated magnetic beads (Invitrogen) and incubated and incubated overnight at 4°C. .. Protein kinase K (20 mg/mL) and 1 μL of 10% SDS in 100 μL of lysis buffer were added to the pellet and incubated at 55°C for 20 min. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated with Trizol reagent (Invitrogen).

    Isolation:

    Article Title: Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD
    Article Snippet: Paragraph title: Biotinylation and isolation of cell surface proteins ... Cell pellets were suspended in 200 μl of lysis buffer containing Protease Inhibitor Mixture (Thermo Scientific).

    Article Title: Piperlongumine regulates epigenetic modulation and alleviates psoriasis-like skin inflammation via inhibition of hyperproliferation and inflammation
    Article Snippet: .. Enzyme-linked immune sorbent assay Skin tissues were homogenized in ice-cold lysis buffer and protein was isolated as per the protocol described previously to determine the various inflammatory cytokines, such as IL-1β, IL-6, IL-17A, IL-22, TGF-β, and TNF-α (Thermo Fisher Scientific, USA). .. The cytokine levels were normalized by the Bradford protein assay.

    Article Title: Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking
    Article Snippet: The genomic DNA was isolated at 36 h post-transfection as described before ( ). .. In brief, the cells were collected by centrifugation and resuspended in lysis buffer consisting of 200 mM NaCl, 10 mM Tris–HCl pH 7.4, 2 mM EDTA pH 8.0, 0.2% (w/v), sodium dodecyl sulphate (SDS) and freshly added proteinase K (Thermo Fisher Scientific; Cat. No.: #EO0491) at a final concentration of 200 ng ml−1 .

    Article Title: MicroRNA-29 family functions as a tumor suppressor by targeting RPS15A and regulating cell cycle in hepatocellular carcinoma
    Article Snippet: .. Protein kinase K (20 mg/mL) and 1 μL of 10% SDS in 100 μL of lysis buffer were added to the pellet and incubated at 55°C for 20 min. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated with Trizol reagent (Invitrogen). .. The levels of RPS15A in the Bio-miR-29a, b, c pull-down were quantified by qRT-PCR.

    Purification:

    Article Title: Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis
    Article Snippet: Cell sorting, reverse transcription, amplification, and sequencing One thousand cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL−1 RNase Inhibitor (Takara). .. The amplified cDNA products were purified with Agencourt XP DNA beads (Beckman), and the concentration of each sample was quantified by Qubit HsDNA kits (Invitrogen).

    Polymerase Chain Reaction:

    Article Title: Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis
    Article Snippet: Cell sorting, reverse transcription, amplification, and sequencing One thousand cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL−1 RNase Inhibitor (Takara). .. Transcriptome amplifications were performed according to Smart-Seq2 protocol [ ] with modification of reagent amount and PCR cycle numbers.

    Protein Extraction:

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
    Article Snippet: .. The beads were washed three times with lysis buffer, and the precipitates were eluted in 30 μl of M-PER mammalian protein extraction reagent (Thermo, Rockford, IL) and 6× loading buffer by boiling at 95°C for 5 min. .. The beads were spun down in a microcentrifuge at full speed at 4°C for 5 min, and the supernatants were subjected to SDS-10% PAGE.

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma). .. Dissected cochleae from wild type mice were also subject to protein extraction using the same buffer and inhibitors.

    Quantitative RT-PCR:

    Article Title: MicroRNA-29 family functions as a tumor suppressor by targeting RPS15A and regulating cell cycle in hepatocellular carcinoma
    Article Snippet: Protein kinase K (20 mg/mL) and 1 μL of 10% SDS in 100 μL of lysis buffer were added to the pellet and incubated at 55°C for 20 min. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated with Trizol reagent (Invitrogen). .. The levels of RPS15A in the Bio-miR-29a, b, c pull-down were quantified by qRT-PCR.

    Bradford Protein Assay:

    Article Title: EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming
    Article Snippet: Co-immunoprecipitation Cellular lysates were prepared by incubating the cells in lysis buffer (10 mM CHAPS, 50 mM Tris-HCL, pH 8, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) containing a protease inhibitor cocktail (Thermo Fisher Scientific) for 20 min at 4 °C, followed by centrifugation at 14,000g for 15 min at 4 °C. .. The protein concentration of the lysates was determined using the Bradford protein assay kit (Bio-Rad) according to the manufacturer’s protocol.

    Article Title: Piperlongumine regulates epigenetic modulation and alleviates psoriasis-like skin inflammation via inhibition of hyperproliferation and inflammation
    Article Snippet: Enzyme-linked immune sorbent assay Skin tissues were homogenized in ice-cold lysis buffer and protein was isolated as per the protocol described previously to determine the various inflammatory cytokines, such as IL-1β, IL-6, IL-17A, IL-22, TGF-β, and TNF-α (Thermo Fisher Scientific, USA). .. The cytokine levels were normalized by the Bradford protein assay.

    FACS:

    Article Title: Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis
    Article Snippet: .. Cell sorting, reverse transcription, amplification, and sequencing One thousand cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL−1 RNase Inhibitor (Takara). .. Transcriptome amplifications were performed according to Smart-Seq2 protocol [ ] with modification of reagent amount and PCR cycle numbers.

    Mouse Assay:

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma). .. Dissected cochleae from wild type mice were also subject to protein extraction using the same buffer and inhibitors.

    Article Title: Sensory Experience Remodels Genome Architecture in Neural Circuit to Drive Motor Learning
    Article Snippet: RNA-Seq Total RNA was extracted from cerebellum, ADCV, or lobule VI of 6–8 week-old mice using Trizol (Thermo Fisher Scientific) according to the manufacturer’s instructions. .. For chromatin or nucleocytoplasmic fractionation, tissues were homogenized in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton-X, RNaseOUT (Thermo Fisher Scientific)) using a syringe and incubated on ice for 10 minutes.

    SDS Page:

    Article Title: EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming
    Article Snippet: Co-immunoprecipitation Cellular lysates were prepared by incubating the cells in lysis buffer (10 mM CHAPS, 50 mM Tris-HCL, pH 8, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) containing a protease inhibitor cocktail (Thermo Fisher Scientific) for 20 min at 4 °C, followed by centrifugation at 14,000g for 15 min at 4 °C. .. The precipitated proteins were eluted from the beads by resuspending the beads in 2× SDS-PAGE loading buffer and boiling for 10 min.

    Article Title: Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD
    Article Snippet: Cell pellets were suspended in 200 μl of lysis buffer containing Protease Inhibitor Mixture (Thermo Scientific). .. The biotin-labeled cell surface proteins were isolated using NeutrAvidin Agarose columns (Thermo Scientific), eluted with SDS-PAGE sample buffer, and analyzed by immunoblotting.

    Co-Immunoprecipitation Assay:

    Article Title: EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming
    Article Snippet: Co-immunoprecipitation Cellular lysates were prepared by incubating the cells in lysis buffer (10 mM CHAPS, 50 mM Tris-HCL, pH 8, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) containing a protease inhibitor cocktail (Thermo Fisher Scientific) for 20 min at 4 °C, followed by centrifugation at 14,000g for 15 min at 4 °C. .. For immunoprecipitation, 500 µg of protein was incubated with 2 µg specific antibodies for 12 h at 4 °C with constant rotation; 60 µl of 50% protein A or G agarose beads was then added and the incubation was continued for an additional 2 h. Beads were then washed five times using CHAPS co-IP lysis buffer.

    Sample Prep:

    Article Title: Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking
    Article Snippet: Paragraph title: Orthogonal HTGTS sample preparation ... In brief, the cells were collected by centrifugation and resuspended in lysis buffer consisting of 200 mM NaCl, 10 mM Tris–HCl pH 7.4, 2 mM EDTA pH 8.0, 0.2% (w/v), sodium dodecyl sulphate (SDS) and freshly added proteinase K (Thermo Fisher Scientific; Cat. No.: #EO0491) at a final concentration of 200 ng ml−1 .

    Concentration Assay:

    Article Title: Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis
    Article Snippet: Cell sorting, reverse transcription, amplification, and sequencing One thousand cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL−1 RNase Inhibitor (Takara). .. The amplified cDNA products were purified with Agencourt XP DNA beads (Beckman), and the concentration of each sample was quantified by Qubit HsDNA kits (Invitrogen).

    Article Title: Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking
    Article Snippet: .. In brief, the cells were collected by centrifugation and resuspended in lysis buffer consisting of 200 mM NaCl, 10 mM Tris–HCl pH 7.4, 2 mM EDTA pH 8.0, 0.2% (w/v), sodium dodecyl sulphate (SDS) and freshly added proteinase K (Thermo Fisher Scientific; Cat. No.: #EO0491) at a final concentration of 200 ng ml−1 . .. After an overnight incubation period at 56°C, the DNA was precipitated by adding isopropanol (1:1) and immediate mixing of the aqueous and organic phases.

    Construct:

    Article Title: Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis
    Article Snippet: Cell sorting, reverse transcription, amplification, and sequencing One thousand cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL−1 RNase Inhibitor (Takara). .. Libraries were constructed and amplified using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Biotech).

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Seventy percent confluent HEK293 cell monolayers cultured on a 10 cm2 dishes were transfected with the RIPOR2-HA construct using Lipofectamine 3000 (Invitrogen) following the manufacturer’s standard instructions. .. Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma).

    Fractionation:

    Article Title: Sensory Experience Remodels Genome Architecture in Neural Circuit to Drive Motor Learning
    Article Snippet: .. For chromatin or nucleocytoplasmic fractionation, tissues were homogenized in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton-X, RNaseOUT (Thermo Fisher Scientific)) using a syringe and incubated on ice for 10 minutes. .. After spinning down, the RNA in the supernatant (nucleocytoplasmic fraction) was extracted with Trizol LS (Thermo Fisher Scientific).

    Molecular Weight:

    Article Title: The effect of NAMPT deletion in projection neurons on the function and structure of neuromuscular junction (NMJ) in mice
    Article Snippet: Briefly, the fresh muscle tissues were dissolved in lysis buffer mixed with protease inhibitor (Cat. No. 36978, Pierce Biotechnology, Rockford, IL) and cocktails of phosphatase inhibitor (Cat. No. P8340 Sigma-Aldrich), and homogenized. .. The protein samples were boiled for 5 minutes, loaded in 10% SDS-polyacrylamide gels, subjected to electrophoresis at 100 mV for 100–110 minutes, and transferred to polyvinylidene fluoride membranes using Mixed Molecular Weight function of Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA).

    Lysis:

    Article Title: EGFL9 promotes breast cancer metastasis by inducing cMET activation and metabolic reprogramming
    Article Snippet: .. Co-immunoprecipitation Cellular lysates were prepared by incubating the cells in lysis buffer (10 mM CHAPS, 50 mM Tris-HCL, pH 8, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) containing a protease inhibitor cocktail (Thermo Fisher Scientific) for 20 min at 4 °C, followed by centrifugation at 14,000g for 15 min at 4 °C. .. The protein concentration of the lysates was determined using the Bradford protein assay kit (Bio-Rad) according to the manufacturer’s protocol.

    Article Title: Core gene insertion in hepatitis B virus genotype G functions at both the encoded amino acid sequence and RNA structure levels to stimulate core protein expression
    Article Snippet: .. Cells were lysed in 100μl of lysis buffer (10 mM HEPES, pH 7.5; 100 mM NaCl; 1 mM EDTA and 1% NP40), and protein concentration in cell lysate was quantified by Pierce™ BCA Protein Assay Kit (Thermo Scientific). .. Core and envelope proteins in cell lysate were detected by Western blot analysis.

    Article Title: Computational and Biochemical Studies of Isothiocyanates as Inhibitors of Proteasomal Cysteine Deubiquitinases in Human Cancer Cells
    Article Snippet: .. The lysis buffer was prepared using 1 mM Tris-HCL (pH 8.0), 1M NaCl, 10% NP-40, 0.5M EDTA in distilled water, and Halt™ protease and phosphatase inhibitor cocktail (Thermo Scientific) was added to the whole lysis buffer in 1:100 ratio. ..

    Article Title: Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD
    Article Snippet: .. Cell pellets were suspended in 200 μl of lysis buffer containing Protease Inhibitor Mixture (Thermo Scientific). .. Cell lysates were collected after centrifugation at 10,000 × g for 2 min at 4 °C.

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
    Article Snippet: .. The beads were washed three times with lysis buffer, and the precipitates were eluted in 30 μl of M-PER mammalian protein extraction reagent (Thermo, Rockford, IL) and 6× loading buffer by boiling at 95°C for 5 min. .. The beads were spun down in a microcentrifuge at full speed at 4°C for 5 min, and the supernatants were subjected to SDS-10% PAGE.

    Article Title: Excised linear introns regulate growth in yeast
    Article Snippet: .. Crude lysates were prepared by re-suspending an aliquot of thawed lysate powder (500–800 μL of loosely packed powder) in 1 mL of lysis buffer (10 mM Tris-HCl [pH 7.4], 5 mM MgCl2 , 100 mM KCl, 1% Triton X−100, 1% Sodium Deoxycholate, 2 mM DTT, 20 U/ml SUPERase•In [Ambion], cOmplete EDTA-free Protease Inhibitor Cocktail [Roche]). ..

    Article Title: Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis
    Article Snippet: .. Cell sorting, reverse transcription, amplification, and sequencing One thousand cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL−1 RNase Inhibitor (Takara). .. Transcriptome amplifications were performed according to Smart-Seq2 protocol [ ] with modification of reagent amount and PCR cycle numbers.

    Article Title: The effect of NAMPT deletion in projection neurons on the function and structure of neuromuscular junction (NMJ) in mice
    Article Snippet: .. Briefly, the fresh muscle tissues were dissolved in lysis buffer mixed with protease inhibitor (Cat. No. 36978, Pierce Biotechnology, Rockford, IL) and cocktails of phosphatase inhibitor (Cat. No. P8340 Sigma-Aldrich), and homogenized. .. The protein concentration of the cell lysate was measured by bicinchoninic acid (BCA) protein assay kit (Cat. No. 23277, Pierce Biotechnology).

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: .. Forty-eight hours post-transfection cells were washed with PBS and proteins were extracted with cold lysis buffer (Pierce) containing a protein inhibitor mixture (Sigma). .. Dissected cochleae from wild type mice were also subject to protein extraction using the same buffer and inhibitors.

    Article Title: Membralin deficiency dysregulates astrocytic glutamate homeostasis, leading to ALS-like impairment
    Article Snippet: .. Spinal cord or astrocyte samples were homogenized in cold lysis buffer comprising 1% NP40, 1% digitonin, 50 mM Tris-HCl (pH 8.0), and 150 mM NaCl supplemented with Halt Protease and Phosphatase Inhibitor Cocktails (Thermo Fisher Scientific). .. Protein concentrations were determined by bicinchoninic (BCA) assay, whereby protein samples were subjected to immunoblot analysis.

    Article Title: Piperlongumine regulates epigenetic modulation and alleviates psoriasis-like skin inflammation via inhibition of hyperproliferation and inflammation
    Article Snippet: .. Enzyme-linked immune sorbent assay Skin tissues were homogenized in ice-cold lysis buffer and protein was isolated as per the protocol described previously to determine the various inflammatory cytokines, such as IL-1β, IL-6, IL-17A, IL-22, TGF-β, and TNF-α (Thermo Fisher Scientific, USA). .. The cytokine levels were normalized by the Bradford protein assay.

    Article Title: Sensory Experience Remodels Genome Architecture in Neural Circuit to Drive Motor Learning
    Article Snippet: .. For chromatin or nucleocytoplasmic fractionation, tissues were homogenized in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton-X, RNaseOUT (Thermo Fisher Scientific)) using a syringe and incubated on ice for 10 minutes. .. After spinning down, the RNA in the supernatant (nucleocytoplasmic fraction) was extracted with Trizol LS (Thermo Fisher Scientific).

    Article Title: Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling
    Article Snippet: .. For cellular protein expression determination, cells were scraped into ice-cold lysis buffer containing 50 mM Tris·HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and protease and phosphatase inhibitors (Halt 78447; Thermo Fisher Scientific). .. The lysates were then triturated through a small-bore needle using a syringe, sonicated, and kept on ice.

    Article Title: Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking
    Article Snippet: .. In brief, the cells were collected by centrifugation and resuspended in lysis buffer consisting of 200 mM NaCl, 10 mM Tris–HCl pH 7.4, 2 mM EDTA pH 8.0, 0.2% (w/v), sodium dodecyl sulphate (SDS) and freshly added proteinase K (Thermo Fisher Scientific; Cat. No.: #EO0491) at a final concentration of 200 ng ml−1 . .. After an overnight incubation period at 56°C, the DNA was precipitated by adding isopropanol (1:1) and immediate mixing of the aqueous and organic phases.

    Article Title: MicroRNA-29 family functions as a tumor suppressor by targeting RPS15A and regulating cell cycle in hepatocellular carcinoma
    Article Snippet: .. Protein kinase K (20 mg/mL) and 1 μL of 10% SDS in 100 μL of lysis buffer were added to the pellet and incubated at 55°C for 20 min. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated with Trizol reagent (Invitrogen). .. The levels of RPS15A in the Bio-miR-29a, b, c pull-down were quantified by qRT-PCR.

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  • 99
    Thermo Fisher western blot analysis cell lysis buffer
    GDNF reduces ER stress and consequently ER stress induced apoptosis in human islets. ( a ) Representative <t>western</t> <t>blot</t> of IRE1α and BiP in human islets treated with Tg (1 µm) with or without GDNF (200 ng/ml) for 48 hrs. ( b , c ) Quantification of IRE1α and BiP band densities normalized to housekeeping gene GAPDH. Data is presented as a fold of vehicle islets, n = 6. ( d ) <t>Cell</t> death <t>analysis</t> by cell death ELISA PLUS of human islets treated with Tg with or without GDNF for 48 hrs, n = 9. ( e ) Representative images showing insulin (red), TUNEL (green) and DAPI (blue) nuclear staining of dispersed human islets treated with Tg with or without GDNF for 48 hrs. ( f ) Score of TUNEL + cells ( g ) and measurement of insulin area to DAPI nuclear staining in dispersed human islets. Data is presented as a fold of vehicle islets. n = 3, five images were taken from each slide and minimum of 2000 cells were scored. ( h ) Representative images showing intact islets stained for PI (red) and FDA (green). For all analysis, data is presented as mean ± SD and p-values were analyzed by nonparametric ANOVA with Dunn’s corrections.*p
    Western Blot Analysis Cell Lysis Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher immunoprecipitation lysis buffer
    HDAC 4 reduces the acetylation and secretion of HMGB 1 in LPS ‐stimulated RAW 264.7 cells and peritoneal macrophages. (A,C) RAW 264.7 cells (A) or peritoneal macrophages (C) were transfected with pc DNA 3.1 (1 μg) or the HDAC 4 vector (1 μg) for 12 h and then treated with LPS (1 μg· mL −1 ) for 12 h. After incubation, cell medium and lysates were collected for measuring HMGB 1 and HDAC 4. Equal volumes of medium were subjected to western blot analysis using an anti‐ HMGB 1 antibody. Cell lysates were subjected to western blot analysis using an anti‐ HDAC 4 antibody. (B,D) RAW 264.7 cells (B) or peritoneal macrophages (D) were transfected with pc DNA 3.1 (1 μg) or the HDAC 4 vector (1 μg) for 12 h prior to LPS (1 μg· mL −1 ) treatment. After incubation for 4 h, cell lysates were subjected to <t>immunoprecipitation</t> using an anti‐ HMGB 1 antibody and then to western blot analysis using an anti‐acetyl‐Lys antibody. The results are expressed as the means ± SEM of three independent experiments. * P
    Immunoprecipitation Lysis Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pierce immunoprecipitation lysis buffer
    C9ORF72–SMCR8–WDR41 form a protein complex. ( A ) Illustration of the gene-editing strategy for making C9ORF72-HA stable expression hES cell lines. Zinc finger nuclease was used to introduce a double-strand break in the AAVS1 locus. The puromycin resistance selection gene and CAGGS -driven C9ORF72-HA expression cassette are stably incorporated into the AAVS1 locus through homologous recombination. ( B ) Identification of protein coimmunoprecipitated with the C9-long protein isoform by quantitative mass spectrometry. The red dots represent proteins significantly enriched in samples with C9-long-HA expression after anti-HA <t>immunoprecipitation</t> with an adjusted P -value
    Pierce Immunoprecipitation Lysis Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    GDNF reduces ER stress and consequently ER stress induced apoptosis in human islets. ( a ) Representative western blot of IRE1α and BiP in human islets treated with Tg (1 µm) with or without GDNF (200 ng/ml) for 48 hrs. ( b , c ) Quantification of IRE1α and BiP band densities normalized to housekeeping gene GAPDH. Data is presented as a fold of vehicle islets, n = 6. ( d ) Cell death analysis by cell death ELISA PLUS of human islets treated with Tg with or without GDNF for 48 hrs, n = 9. ( e ) Representative images showing insulin (red), TUNEL (green) and DAPI (blue) nuclear staining of dispersed human islets treated with Tg with or without GDNF for 48 hrs. ( f ) Score of TUNEL + cells ( g ) and measurement of insulin area to DAPI nuclear staining in dispersed human islets. Data is presented as a fold of vehicle islets. n = 3, five images were taken from each slide and minimum of 2000 cells were scored. ( h ) Representative images showing intact islets stained for PI (red) and FDA (green). For all analysis, data is presented as mean ± SD and p-values were analyzed by nonparametric ANOVA with Dunn’s corrections.*p

    Journal: Scientific Reports

    Article Title: Glial cell-line derived neurotrophic factor protects human islets from nutrient deprivation and endoplasmic reticulum stress induced apoptosis

    doi: 10.1038/s41598-017-01805-1

    Figure Lengend Snippet: GDNF reduces ER stress and consequently ER stress induced apoptosis in human islets. ( a ) Representative western blot of IRE1α and BiP in human islets treated with Tg (1 µm) with or without GDNF (200 ng/ml) for 48 hrs. ( b , c ) Quantification of IRE1α and BiP band densities normalized to housekeeping gene GAPDH. Data is presented as a fold of vehicle islets, n = 6. ( d ) Cell death analysis by cell death ELISA PLUS of human islets treated with Tg with or without GDNF for 48 hrs, n = 9. ( e ) Representative images showing insulin (red), TUNEL (green) and DAPI (blue) nuclear staining of dispersed human islets treated with Tg with or without GDNF for 48 hrs. ( f ) Score of TUNEL + cells ( g ) and measurement of insulin area to DAPI nuclear staining in dispersed human islets. Data is presented as a fold of vehicle islets. n = 3, five images were taken from each slide and minimum of 2000 cells were scored. ( h ) Representative images showing intact islets stained for PI (red) and FDA (green). For all analysis, data is presented as mean ± SD and p-values were analyzed by nonparametric ANOVA with Dunn’s corrections.*p

    Article Snippet: Western blot analysis Cell lysis buffer (RIPA buffer supplemented with Halt protease inhibitor (Thermo scientific, Oslo, Norway) or tissue lysis buffer (RIPA buffer containing halt protease-phosphatase inhibitors and 1% sodium dodecyl sulfate) was added to human islets pellet (100 islets) or frozen graft-bearing kidney samples before proceeding to mechanical disruption using sonication.

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining

    HDAC 4 reduces the acetylation and secretion of HMGB 1 in LPS ‐stimulated RAW 264.7 cells and peritoneal macrophages. (A,C) RAW 264.7 cells (A) or peritoneal macrophages (C) were transfected with pc DNA 3.1 (1 μg) or the HDAC 4 vector (1 μg) for 12 h and then treated with LPS (1 μg· mL −1 ) for 12 h. After incubation, cell medium and lysates were collected for measuring HMGB 1 and HDAC 4. Equal volumes of medium were subjected to western blot analysis using an anti‐ HMGB 1 antibody. Cell lysates were subjected to western blot analysis using an anti‐ HDAC 4 antibody. (B,D) RAW 264.7 cells (B) or peritoneal macrophages (D) were transfected with pc DNA 3.1 (1 μg) or the HDAC 4 vector (1 μg) for 12 h prior to LPS (1 μg· mL −1 ) treatment. After incubation for 4 h, cell lysates were subjected to immunoprecipitation using an anti‐ HMGB 1 antibody and then to western blot analysis using an anti‐acetyl‐Lys antibody. The results are expressed as the means ± SEM of three independent experiments. * P

    Journal: FEBS Open Bio

    Article Title: Degradation of histone deacetylase 4 via the TLR4/ JAK/ STAT1 signaling pathway promotes the acetylation of high mobility group box 1 ( HMGB1) in lipopolysaccharide‐activated macrophages

    doi: 10.1002/2211-5463.12456

    Figure Lengend Snippet: HDAC 4 reduces the acetylation and secretion of HMGB 1 in LPS ‐stimulated RAW 264.7 cells and peritoneal macrophages. (A,C) RAW 264.7 cells (A) or peritoneal macrophages (C) were transfected with pc DNA 3.1 (1 μg) or the HDAC 4 vector (1 μg) for 12 h and then treated with LPS (1 μg· mL −1 ) for 12 h. After incubation, cell medium and lysates were collected for measuring HMGB 1 and HDAC 4. Equal volumes of medium were subjected to western blot analysis using an anti‐ HMGB 1 antibody. Cell lysates were subjected to western blot analysis using an anti‐ HDAC 4 antibody. (B,D) RAW 264.7 cells (B) or peritoneal macrophages (D) were transfected with pc DNA 3.1 (1 μg) or the HDAC 4 vector (1 μg) for 12 h prior to LPS (1 μg· mL −1 ) treatment. After incubation for 4 h, cell lysates were subjected to immunoprecipitation using an anti‐ HMGB 1 antibody and then to western blot analysis using an anti‐acetyl‐Lys antibody. The results are expressed as the means ± SEM of three independent experiments. * P

    Article Snippet: Immunoprecipitation For immunoprecipitation, cells were lysed using immunoprecipitation lysis buffer (Thermo Fisher Scientific).

    Techniques: Transfection, Plasmid Preparation, Incubation, Western Blot, Immunoprecipitation

    Acetylation and secretion of HMGB 1 through TLR 4/ JAK / STAT 1 signaling in LPS ‐activated RAW 264.7 cells. (A,B) Cells were treated with TAK ‐242 (10, 100 or 1000 n m ) (A) or pyridone 6 (1, 10 or 100 n m ) (B) for 30 min prior to LPS (1 μg· mL −1 ) treatment. After incubation for 12 h, medium was collected for the detection of HMGB 1. Equal volumes of medium were subjected to western blot analysis using an anti‐ HMGB 1 antibody. (C,D) Cells were treated with TAK ‐242 (1 μ m ) (C) or pyridone 6 (100 n m ) (D) for 30 min prior to LPS (1 μg· mL −1 ) treatment. After incubation for 4 h, cell lysates were subjected to immunoprecipitation using an anti‐ HMGB 1 antibody and then to western blot analysis using an anti‐acetyl‐Lys antibody. The results are expressed as the means ± SEM of three independent experiments. * P

    Journal: FEBS Open Bio

    Article Title: Degradation of histone deacetylase 4 via the TLR4/ JAK/ STAT1 signaling pathway promotes the acetylation of high mobility group box 1 ( HMGB1) in lipopolysaccharide‐activated macrophages

    doi: 10.1002/2211-5463.12456

    Figure Lengend Snippet: Acetylation and secretion of HMGB 1 through TLR 4/ JAK / STAT 1 signaling in LPS ‐activated RAW 264.7 cells. (A,B) Cells were treated with TAK ‐242 (10, 100 or 1000 n m ) (A) or pyridone 6 (1, 10 or 100 n m ) (B) for 30 min prior to LPS (1 μg· mL −1 ) treatment. After incubation for 12 h, medium was collected for the detection of HMGB 1. Equal volumes of medium were subjected to western blot analysis using an anti‐ HMGB 1 antibody. (C,D) Cells were treated with TAK ‐242 (1 μ m ) (C) or pyridone 6 (100 n m ) (D) for 30 min prior to LPS (1 μg· mL −1 ) treatment. After incubation for 4 h, cell lysates were subjected to immunoprecipitation using an anti‐ HMGB 1 antibody and then to western blot analysis using an anti‐acetyl‐Lys antibody. The results are expressed as the means ± SEM of three independent experiments. * P

    Article Snippet: Immunoprecipitation For immunoprecipitation, cells were lysed using immunoprecipitation lysis buffer (Thermo Fisher Scientific).

    Techniques: Incubation, Western Blot, Immunoprecipitation

    SFKs are necessary for mTOR and BCAP phosphorylation in human pDCs. ( a ) CAL-1 cells were pre-treated for 1 h with PP2 (10 μM; blue) or DMSO control (black) before stimulation with R848 for the indicated time periods (minutes). p-MTOR was determined by flow cytometry. Histograms depict the 15 min time point. ( b ) Human PBMCs were pre-treated as in a and stimulated for 15 min with R848. p-MTOR was determined by flow cytometry in gated pDCs. Lines in graph connect the same donor and the histogram depicts a representative donor; dotted lines, unstimulated; solid lines, R848 stimulation. ( c ) CAL-1 cells were pre-treated as in a and then stimulated with R848 for 15 min. Protein immunoprecipitation was performed using anti-BCAP antibody or isotype control antibody and levels of tyrosine phosphorylation (p-Tyr) and BCAP were assessed by immunoblot. ( d ) CAL-1 cells were pre-treated as in a and then stimulated with R848 for the indicated time periods (minutes). p-AKT Ser473 and AKT levels were determined by immunoblot. Data are representative of two ( d ) or three ( a , c ) independent experiments or five donors processed separately ( b ). Graphs depict mean±s.d. of replicates within one representative experiment ( a , c , d ) or individual donors ( b ). Two-way ANOVA ( a ) and two-ways Student's t -test ( b ) were used for statistical analyses. * P

    Journal: Nature Communications

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses

    doi: 10.1038/ncomms14830

    Figure Lengend Snippet: SFKs are necessary for mTOR and BCAP phosphorylation in human pDCs. ( a ) CAL-1 cells were pre-treated for 1 h with PP2 (10 μM; blue) or DMSO control (black) before stimulation with R848 for the indicated time periods (minutes). p-MTOR was determined by flow cytometry. Histograms depict the 15 min time point. ( b ) Human PBMCs were pre-treated as in a and stimulated for 15 min with R848. p-MTOR was determined by flow cytometry in gated pDCs. Lines in graph connect the same donor and the histogram depicts a representative donor; dotted lines, unstimulated; solid lines, R848 stimulation. ( c ) CAL-1 cells were pre-treated as in a and then stimulated with R848 for 15 min. Protein immunoprecipitation was performed using anti-BCAP antibody or isotype control antibody and levels of tyrosine phosphorylation (p-Tyr) and BCAP were assessed by immunoblot. ( d ) CAL-1 cells were pre-treated as in a and then stimulated with R848 for the indicated time periods (minutes). p-AKT Ser473 and AKT levels were determined by immunoblot. Data are representative of two ( d ) or three ( a , c ) independent experiments or five donors processed separately ( b ). Graphs depict mean±s.d. of replicates within one representative experiment ( a , c , d ) or individual donors ( b ). Two-way ANOVA ( a ) and two-ways Student's t -test ( b ) were used for statistical analyses. * P

    Article Snippet: Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation.

    Techniques: Flow Cytometry, Cytometry, Immunoprecipitation

    pDCs have constitutive LYN and FYN expression and activating phosphorylation. The expression of SFK mRNAs in mouse immune subsets ( a ) and human cell lines ( b ) were assessed by qPCR and normalized against the expression of GAPDH. In a , the immune subsets were FACS purified from freshly isolated spleen or from BM cells cultivated for 8 days in the presence of Flt3L. In b , cells from human cell lines were collected before reaching confluency and then total RNAs were extracted. Splenic pDCs are highlighted in solid red; BM-derived pDCs are highlighted in black-diagonal striped red; CAL-1 are highlighted in black-horizontal striped red. Graph bars represent mean±s.d. of two independent experiments. A549: lung epithelial cell line; CAL-1: human pDC cell line; Jurkat: T-cell line; Ramos: B-cell line; THP-1: monocyte cell line. ( c , d ) CAL-1 cells ( c ) or human primary pDCs ( d ) were stimulated with R848 for the indicated time periods (minutes) and SFK activating tyrosine phosphorylation (Y416) and GAPDH were assessed by immunoblot. ( e , f ) CAL-1 cells were stimulated with R848 for the indicated time periods (minutes) and protein immunoprecipitation was performed using anti-LYN ( e ) or anti-FYN ( f ) antibodies or isotype control antibody. Levels of LYN or FYN activating tyrosine phosphorylation and total LYN or FYN were assessed by immunoblot. Results are representative of two independent experiments ( c – e , f ) or three individual donors ( d ).

    Journal: Nature Communications

    Article Title: Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses

    doi: 10.1038/ncomms14830

    Figure Lengend Snippet: pDCs have constitutive LYN and FYN expression and activating phosphorylation. The expression of SFK mRNAs in mouse immune subsets ( a ) and human cell lines ( b ) were assessed by qPCR and normalized against the expression of GAPDH. In a , the immune subsets were FACS purified from freshly isolated spleen or from BM cells cultivated for 8 days in the presence of Flt3L. In b , cells from human cell lines were collected before reaching confluency and then total RNAs were extracted. Splenic pDCs are highlighted in solid red; BM-derived pDCs are highlighted in black-diagonal striped red; CAL-1 are highlighted in black-horizontal striped red. Graph bars represent mean±s.d. of two independent experiments. A549: lung epithelial cell line; CAL-1: human pDC cell line; Jurkat: T-cell line; Ramos: B-cell line; THP-1: monocyte cell line. ( c , d ) CAL-1 cells ( c ) or human primary pDCs ( d ) were stimulated with R848 for the indicated time periods (minutes) and SFK activating tyrosine phosphorylation (Y416) and GAPDH were assessed by immunoblot. ( e , f ) CAL-1 cells were stimulated with R848 for the indicated time periods (minutes) and protein immunoprecipitation was performed using anti-LYN ( e ) or anti-FYN ( f ) antibodies or isotype control antibody. Levels of LYN or FYN activating tyrosine phosphorylation and total LYN or FYN were assessed by immunoblot. Results are representative of two independent experiments ( c – e , f ) or three individual donors ( d ).

    Article Snippet: Immunoprecipitation and immunoblotting CAL-1 cells were pre-treated with either PP2 or DMSO control for 1 h, stimulated with R848 (1 μg ml−1 ) and subsequently lysed with immunoprecipitation lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail set III (EMD Millipore) and phosphatase inhibitor cocktail set I and II (EMD Millipore) for immunoprecipitation analysis, with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail set III (EMD Millipore) for immunoblotting analysis or processed with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific) for subcellular fractionation.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, FACS, Purification, Isolation, Derivative Assay, Immunoprecipitation

    C9ORF72–SMCR8–WDR41 form a protein complex. ( A ) Illustration of the gene-editing strategy for making C9ORF72-HA stable expression hES cell lines. Zinc finger nuclease was used to introduce a double-strand break in the AAVS1 locus. The puromycin resistance selection gene and CAGGS -driven C9ORF72-HA expression cassette are stably incorporated into the AAVS1 locus through homologous recombination. ( B ) Identification of protein coimmunoprecipitated with the C9-long protein isoform by quantitative mass spectrometry. The red dots represent proteins significantly enriched in samples with C9-long-HA expression after anti-HA immunoprecipitation with an adjusted P -value

    Journal: Genes & Development

    Article Title: The C9orf72-interacting protein Smcr8 is a negative regulator of autoimmunity and lysosomal exocytosis

    doi: 10.1101/gad.313932.118

    Figure Lengend Snippet: C9ORF72–SMCR8–WDR41 form a protein complex. ( A ) Illustration of the gene-editing strategy for making C9ORF72-HA stable expression hES cell lines. Zinc finger nuclease was used to introduce a double-strand break in the AAVS1 locus. The puromycin resistance selection gene and CAGGS -driven C9ORF72-HA expression cassette are stably incorporated into the AAVS1 locus through homologous recombination. ( B ) Identification of protein coimmunoprecipitated with the C9-long protein isoform by quantitative mass spectrometry. The red dots represent proteins significantly enriched in samples with C9-long-HA expression after anti-HA immunoprecipitation with an adjusted P -value

    Article Snippet: Cells were washed on the plate with PBS and lysed in Pierce immunoprecipitation lysis buffer (Thermo Fisher Scientific, PI87787) supplemented with Pierce Halt protease and phosphatase inhibitor cocktail (no. 78443).

    Techniques: Expressing, Zinc-Fingers, Introduce, Selection, Stable Transfection, Homologous Recombination, Mass Spectrometry, Immunoprecipitation