cell lysis buffer  (Thermo Fisher)


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    Name:
    Lysis Buffer A
    Description:
    Thermo Scientific GeneJET Lysis Buffer A is a component of the GeneJET Plant Genomic DNAPurification Mini Kit K0791 K0792 and may be purchased separately
    Catalog Number:
    r1661
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|DNA Extraction|Genomic DNA Purification|gDNA from Plant & Fungi
    Category:
    Lab Reagents and Chemicals
    Buy from Supplier


    Structured Review

    Thermo Fisher cell lysis buffer
    Thermo Scientific GeneJET Lysis Buffer A is a component of the GeneJET Plant Genomic DNAPurification Mini Kit K0791 K0792 and may be purchased separately
    https://www.bioz.com/result/cell lysis buffer/product/Thermo Fisher
    Average 99 stars, based on 598 article reviews
    Price from $9.99 to $1999.99
    cell lysis buffer - by Bioz Stars, 2020-09
    99/100 stars

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    Related Articles

    Sonication:

    Article Title: The WblC/WhiB7 Transcription Factor Controls Intrinsic Resistance to Translation-Targeting Antibiotics by Altering Ribosome Composition
    Article Snippet: .. Then, the cells were harvested and washed with wash buffer (20 mM Tris-Cl [pH 7.4], 100 mM NaCl, 10 mM MgCl2 ) and were lysed by sonication in lysis buffer (20 mM Tris-Cl [pH 7.4], 200 mM NH4 Cl, 10 mM MgCl2 , 0.5 mM EDTA, 6 mM β-mercaptoethanol) with 5 U/ml RNase inhibitor (Applied Biosystems) and 1 mM phenylmethylsulfonyl fluoride (PMSF). .. After 5 U/ml Turbo DNase (Ambion) treatment at 4°C for 15 min, lysates were clarified twice by centrifugation (22,000 × g, 10 min, 4°C).

    Proximity Ligation Assay:

    Article Title: Identification of inhibitors regulating cell proliferation and FUS-DDIT3 expression in myxoid liposarcoma using combined DNA, mRNA, and protein analyses
    Article Snippet: .. Therefore, to test how efficiently we could quantify FUS-DDIT3 and FUS we also tested a standard PLA lysis buffer (Protein Quant Sample Lysis Buffer, Applied Biosystems). .. Supplementary Figure shows that the BSA direct lysis buffer is superior the PLA lysis buffer to quantify DNA, while they were equally suitable for quantifying mRNA.

    Isolation:

    Article Title: Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells
    Article Snippet: .. Real time-qPCR analysis Organoids were lysed in lysis buffer and mRNA was isolated from organoids using the Ambion mRNA Isolation Kit (Life Technologies) according to the manufacturer's instructions. .. We converted 0.5-1 µg mRNA to cDNA in the presence of thermostable RNAse inhibitor and a reaction mix containing GoScript reverse transcriptase (Promega), MgCl2 , nucleotide mix and 5× reaction buffers, using standard GoScript reverse transcription protocol.

    Article Title: Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion
    Article Snippet: .. Gene expression by microarray and qPCR For microarray, lung macrophages (10,000 cells) were directly sorted into RNA lysis buffer (Ambion RNAqueous Micro for RNA isolation) and frozen on dry ice. .. RNA was isolated, amplified using the AminoAllyl MessageAmp II kit (Life Technologies, Grand Island, NY), coupled to Cy3, and hybridized to SurePrint Mouse 8x60k gene expression microarrays (Agilent Technologies, Santa Clara CA) as per manufacturer recommendations.

    Article Title: The long non-coding RNA Meg3 is dispensable for hematopoietic stem cells
    Article Snippet: .. Gene Expression Analysis by qPCR HSCs (MxCre or MxCre Meg3mat flox/pat wt LSK CD150+ CD48− CD34− or fetal liver HSCs: embryonic stage 13.5 CD48− CD41+ cKit+ CD34+; adult HSCs (8–12 weeks): LSK CD150+ CD48− CD34− CD135−; aged HSCs (24 month): LSK CD150+ CD48− CD34− CD135−) were FACS sorted into RNA lysis buffer (ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA)) for qPCR analysis and stored at −80 °C. .. RNA was isolated using the ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA) following manufacturer’s instructions.

    Real-time Polymerase Chain Reaction:

    Article Title: Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion
    Article Snippet: .. Gene expression by microarray and qPCR For microarray, lung macrophages (10,000 cells) were directly sorted into RNA lysis buffer (Ambion RNAqueous Micro for RNA isolation) and frozen on dry ice. .. RNA was isolated, amplified using the AminoAllyl MessageAmp II kit (Life Technologies, Grand Island, NY), coupled to Cy3, and hybridized to SurePrint Mouse 8x60k gene expression microarrays (Agilent Technologies, Santa Clara CA) as per manufacturer recommendations.

    Article Title: The long non-coding RNA Meg3 is dispensable for hematopoietic stem cells
    Article Snippet: .. Gene Expression Analysis by qPCR HSCs (MxCre or MxCre Meg3mat flox/pat wt LSK CD150+ CD48− CD34− or fetal liver HSCs: embryonic stage 13.5 CD48− CD41+ cKit+ CD34+; adult HSCs (8–12 weeks): LSK CD150+ CD48− CD34− CD135−; aged HSCs (24 month): LSK CD150+ CD48− CD34− CD135−) were FACS sorted into RNA lysis buffer (ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA)) for qPCR analysis and stored at −80 °C. .. RNA was isolated using the ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA) following manufacturer’s instructions.

    Microarray:

    Article Title: Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion
    Article Snippet: .. Gene expression by microarray and qPCR For microarray, lung macrophages (10,000 cells) were directly sorted into RNA lysis buffer (Ambion RNAqueous Micro for RNA isolation) and frozen on dry ice. .. RNA was isolated, amplified using the AminoAllyl MessageAmp II kit (Life Technologies, Grand Island, NY), coupled to Cy3, and hybridized to SurePrint Mouse 8x60k gene expression microarrays (Agilent Technologies, Santa Clara CA) as per manufacturer recommendations.

    Incubation:

    Article Title: Functional Mapping of Transcription Factor Grf10 That Regulates Adenine-Responsive and Filamentation Genes in Candida albicans
    Article Snippet: .. Briefly, the frozen cell pellet was resuspended in 200 µl of lysis buffer (0.1 M NaOH, 0.5 M EDTA, 2% SDS, 2% β-mercaptoethanol, protease inhibitor cocktail mix [Thermo Scientific]), and incubated for 10 min at 90°C. ..

    Expressing:

    Article Title: Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion
    Article Snippet: .. Gene expression by microarray and qPCR For microarray, lung macrophages (10,000 cells) were directly sorted into RNA lysis buffer (Ambion RNAqueous Micro for RNA isolation) and frozen on dry ice. .. RNA was isolated, amplified using the AminoAllyl MessageAmp II kit (Life Technologies, Grand Island, NY), coupled to Cy3, and hybridized to SurePrint Mouse 8x60k gene expression microarrays (Agilent Technologies, Santa Clara CA) as per manufacturer recommendations.

    Article Title: The long non-coding RNA Meg3 is dispensable for hematopoietic stem cells
    Article Snippet: .. Gene Expression Analysis by qPCR HSCs (MxCre or MxCre Meg3mat flox/pat wt LSK CD150+ CD48− CD34− or fetal liver HSCs: embryonic stage 13.5 CD48− CD41+ cKit+ CD34+; adult HSCs (8–12 weeks): LSK CD150+ CD48− CD34− CD135−; aged HSCs (24 month): LSK CD150+ CD48− CD34− CD135−) were FACS sorted into RNA lysis buffer (ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA)) for qPCR analysis and stored at −80 °C. .. RNA was isolated using the ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA) following manufacturer’s instructions.

    Protease Inhibitor:

    Article Title: Functional Mapping of Transcription Factor Grf10 That Regulates Adenine-Responsive and Filamentation Genes in Candida albicans
    Article Snippet: .. Briefly, the frozen cell pellet was resuspended in 200 µl of lysis buffer (0.1 M NaOH, 0.5 M EDTA, 2% SDS, 2% β-mercaptoethanol, protease inhibitor cocktail mix [Thermo Scientific]), and incubated for 10 min at 90°C. ..

    FACS:

    Article Title: The long non-coding RNA Meg3 is dispensable for hematopoietic stem cells
    Article Snippet: .. Gene Expression Analysis by qPCR HSCs (MxCre or MxCre Meg3mat flox/pat wt LSK CD150+ CD48− CD34− or fetal liver HSCs: embryonic stage 13.5 CD48− CD41+ cKit+ CD34+; adult HSCs (8–12 weeks): LSK CD150+ CD48− CD34− CD135−; aged HSCs (24 month): LSK CD150+ CD48− CD34− CD135−) were FACS sorted into RNA lysis buffer (ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA)) for qPCR analysis and stored at −80 °C. .. RNA was isolated using the ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA) following manufacturer’s instructions.

    Lysis:

    Article Title: Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells
    Article Snippet: .. Real time-qPCR analysis Organoids were lysed in lysis buffer and mRNA was isolated from organoids using the Ambion mRNA Isolation Kit (Life Technologies) according to the manufacturer's instructions. .. We converted 0.5-1 µg mRNA to cDNA in the presence of thermostable RNAse inhibitor and a reaction mix containing GoScript reverse transcriptase (Promega), MgCl2 , nucleotide mix and 5× reaction buffers, using standard GoScript reverse transcription protocol.

    Article Title: Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion
    Article Snippet: .. Gene expression by microarray and qPCR For microarray, lung macrophages (10,000 cells) were directly sorted into RNA lysis buffer (Ambion RNAqueous Micro for RNA isolation) and frozen on dry ice. .. RNA was isolated, amplified using the AminoAllyl MessageAmp II kit (Life Technologies, Grand Island, NY), coupled to Cy3, and hybridized to SurePrint Mouse 8x60k gene expression microarrays (Agilent Technologies, Santa Clara CA) as per manufacturer recommendations.

    Article Title: Characterisation of immune cell function in fragment and full-length Huntington's disease mouse models
    Article Snippet: .. Blood monocytes Blood was obtained by cardiac puncture, collected into EDTA coated tubes (BD Vacutainer), and 1 ml red blood cell lysis buffer (ammonium chloride buffer; eBioscience) was added per 1 ml of blood. .. Following centrifugation at 300 × g for 5 min, the lysis step was repeated twice.

    Article Title: The WblC/WhiB7 Transcription Factor Controls Intrinsic Resistance to Translation-Targeting Antibiotics by Altering Ribosome Composition
    Article Snippet: .. Then, the cells were harvested and washed with wash buffer (20 mM Tris-Cl [pH 7.4], 100 mM NaCl, 10 mM MgCl2 ) and were lysed by sonication in lysis buffer (20 mM Tris-Cl [pH 7.4], 200 mM NH4 Cl, 10 mM MgCl2 , 0.5 mM EDTA, 6 mM β-mercaptoethanol) with 5 U/ml RNase inhibitor (Applied Biosystems) and 1 mM phenylmethylsulfonyl fluoride (PMSF). .. After 5 U/ml Turbo DNase (Ambion) treatment at 4°C for 15 min, lysates were clarified twice by centrifugation (22,000 × g, 10 min, 4°C).

    Article Title: Functional Mapping of Transcription Factor Grf10 That Regulates Adenine-Responsive and Filamentation Genes in Candida albicans
    Article Snippet: .. Briefly, the frozen cell pellet was resuspended in 200 µl of lysis buffer (0.1 M NaOH, 0.5 M EDTA, 2% SDS, 2% β-mercaptoethanol, protease inhibitor cocktail mix [Thermo Scientific]), and incubated for 10 min at 90°C. ..

    Article Title: The long non-coding RNA Meg3 is dispensable for hematopoietic stem cells
    Article Snippet: .. Gene Expression Analysis by qPCR HSCs (MxCre or MxCre Meg3mat flox/pat wt LSK CD150+ CD48− CD34− or fetal liver HSCs: embryonic stage 13.5 CD48− CD41+ cKit+ CD34+; adult HSCs (8–12 weeks): LSK CD150+ CD48− CD34− CD135−; aged HSCs (24 month): LSK CD150+ CD48− CD34− CD135−) were FACS sorted into RNA lysis buffer (ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA)) for qPCR analysis and stored at −80 °C. .. RNA was isolated using the ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen, Carlsbad, CA, USA) following manufacturer’s instructions.

    Article Title: Identification of inhibitors regulating cell proliferation and FUS-DDIT3 expression in myxoid liposarcoma using combined DNA, mRNA, and protein analyses
    Article Snippet: .. Therefore, to test how efficiently we could quantify FUS-DDIT3 and FUS we also tested a standard PLA lysis buffer (Protein Quant Sample Lysis Buffer, Applied Biosystems). .. Supplementary Figure shows that the BSA direct lysis buffer is superior the PLA lysis buffer to quantify DNA, while they were equally suitable for quantifying mRNA.

    Article Title: H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis
    Article Snippet: .. Treated HACs and MSCs were washed twice in PBS, and cells were harvested in cells to complementary DNA (cDNA) lysis buffer (Ambion; Thermo Fisher Scientific, Austin, TX, USA) prior to cDNA synthesis. ..

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    Thermo Fisher ripa buffer lyse
    Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were <t>G2</t> (included in the EZ1 Tissue kit) and Pierce <t>RIPA</t> Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.
    Ripa Buffer Lyse, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ripa buffer lyse/product/Thermo Fisher
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ripa buffer lyse - by Bioz Stars, 2020-09
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    85
    Thermo Fisher mild immunoprecipitation buffer
    Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, <t>immunoprecipitation;</t> FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.
    Mild Immunoprecipitation Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mild immunoprecipitation buffer/product/Thermo Fisher
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    mild immunoprecipitation buffer - by Bioz Stars, 2020-09
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    86
    Thermo Fisher bm pbs mixture
    Scheme of BMSC isolation. <t>BM-PBS</t> aspirate was divided into four fractions for comparative isolation of <t>BMSCs:</t> 1) untreated whole BM aspirate, 2) 3 volumes of RBC lysis with ammonium chloride, 3) 6 volumes of RBC lysis, or 4) Ficoll density-gradient centrifugation. Finally, BM-PBS mixtures were added to the cell culture medium.
    Bm Pbs Mixture, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bm pbs mixture/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bm pbs mixture - by Bioz Stars, 2020-09
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    Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.

    Journal: Scientific Reports

    Article Title: Plasmid-normalized quantification of relative mitochondrial DNA copy number

    doi: 10.1038/s41598-018-33684-5

    Figure Lengend Snippet: Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.

    Article Snippet: After centrifugation (2 min at 2000 × g) the white blood cell pellet obtained was resuspended using two different buffers: the G2 buffer included in the kit and a Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Incubation, Lysis

    Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, immunoprecipitation; FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.

    Journal: The Journal of Cell Biology

    Article Title: Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

    doi: 10.1083/jcb.201105101

    Figure Lengend Snippet: Hectd1 functions as a Ub ligase. (A) Diagrammatic sketch of the Hectd1 alleles and plasmid constructs used in this study: Hectd1 W (W; wild type), Hectd1 O (O; opm ; openmind ), and Hectd1 X (X; XC gene trap) are mouse alleles; Myc-Hectd1 ANK , HA-Hectd1, and HA-Hectd1* are mammalian expression constructs. Hectd1 O was generated in an ENU mutagenesis screen and harbors a missense mutation resulting in truncation of Hectd1. Hectd1 X is a gene trap allele where the Ub ligase domain is disrupted by insertion of a β-geo (LacZ) cassette ( Zohn et al., 2007 ). HA-Hectd1 ANK consists of amino acids 1–551 of Hectd1 encompassing the ankyrin (ANK) domain. pCMVHA-Hectd1* is Ub ligase deficient because of mutation of the active site cysteine (C2579G). Other motifs present in Hectd1 include Mindbomb (mib) and Sad1/UNC (SUN) domains. The inverted Y denotes the paratope of the Hectd1 antibody that recognizes Hectd1 W and Hectd1 X but not Hectd1 O proteins. (B) Reduced ubiquitination of Hectd1 and associated proteins in HEK293T cells expressing cysteine mutant Hectd1*. HA-Hectd1 immunoprecipitates were subjected to Western blot analyses to detect Hectd1 and mono- and polyubiquitinylated protein conjugates (FK2). (C) The appearance of HMW Hectd1 is dependent on its ligase activity in vivo. Hectd1 immunoprecipitates from E11.5 embryo heads of the indicated genotypes were probed with anti-Hectd1 antibody. (D) The conjugation of K63-linked Ub chains onto Hectd1 is dependent on its ligase activity. Hectd1 was immunoprecipitated from Hectd1 W and Hectd1 X CM cultures and immunoblotted with antibodies to detect K63-linked Ub chains (K63Ub) and Hectd1. (E and F) Reduction of total Ub proteins in Hectd1 X compared with Hectd1 W CM cultures. (E) Ub proteins were pulled down (PD) using Rad23 beads followed by immunoblotting to detect mono- and polyubiquitinylated protein conjugates (FK2). (F) Quantitation of normalized intensity of Western blots shown in E. Error bars represent the mean ± SEM of two independent experiments performed in triplicate. Statistical significance was determined by paired two-tailed Student’s t tests. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; Con, control; IB, immunoblot; IP, immunoprecipitation; FK2, anti–mono- and polyubiquitinylated proteins; (Ub) n , Ub n ; K63Ub, lysine 63 linked Ub n chains.

    Article Snippet: For lenient binding conditions, a mild immunoprecipitation buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 with protease inhibitor cocktail) was used, and for stringent binding conditions, radioimmunoprecipitation assay buffer (no. 89901; Thermo Fisher Scientific) with protease inhibition cocktail (Roche) was used.

    Techniques: Plasmid Preparation, Construct, Expressing, Generated, Mutagenesis, Western Blot, Activity Assay, In Vivo, Conjugation Assay, Immunoprecipitation, Quantitation Assay, Two Tailed Test

    Hectd1 physically interacts with Hsp90α. (A) Yeast two-hybrid screening of an E11.5 embryonic mouse cDNA library using Hectd1 ANK as bait detected Hsp90α (Gene ID: Hsp90aa1). Two identical clones of Hsp90α consisting of amino acids 241–459 (Hsp90αbd) of the 732–amino acid Hsp90α protein partially overlap with the first charged domain (CD1, amino acids 236–272) and the ATPase domain (amino acids 272–618) of Hsp90α. Black dots indicate locations of Hsp90 peptide fragments found by LC-MS analysis: NPDDITQEEYGEFYK (300–315), TLTIVDTGIGMTK (88–100), KADLINNLGTIAKS (100–113), and GVVDSEDLPLNISR (387–400). Peptides common to Hsp90β include: KEDQTEYLEERR (190–201), RDNSTMGYMMAKK (620–632), and YIDQEELNK (284–292). The C-terminal amino acid sequence MEEVD of Hsp90α is essential for regulated secretion. (B and C) Liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomic screening of Hectd1-binding proteins from E10.5 Hectd1 W embryo head lysates. Hectd1 was immunoprecipitated and associated proteins were resolved by 3–8% Tris-Acetate SDS-PAGE and visualized by Coomassie staining. (B) Individual bands from the Coomassie-stained gel were subjected to tryptic proteolysis, and the resulting peptides were analyzed by LC-MS/MS. (C) Representative MS/MS of a 1,293.54 D peptide. This and six other peptides (dots in A) with high XC scores (3.3–3.7) were identified as belonging to Hsp90α and Hsp90β when searched against the mouse Uniprot protein database using the Sequest algorithm as diagrammed in A. (D) Hsp90αbd binds to Hectd1 ANK in rabbit reticulocyte lysates. In vitro translated, biotinylated Hsp90αbd and Hectd1 ANK were bound and immunoprecipitated using the indicated antibodies and detected by Western blotting with streptavidin-HRP. (E and F) Hectd1 binds to Hsp90 in HEK293T cells. Cells were transfected and immunoprecipitated proteins were subjected to Western blot analyses as indicated. (E) Hsp90αbd binds to Hectd1 ANK in HEK293T cells. (F) Full-length Hsp90 and Hectd1 bind in HEK293T cells. (G) Hsp90 binds to Hectd1 in the developing embryo. Hectd1 was immunoprecipitated from lysates prepared from E12.5 Hectd1 W and Hectd1 O embryo heads and subjected to Western blot analysis as indicated ( n = 4). (H and I) Hsp90 binds to Hectd1 but not the related HECT domain containing Nedd4 Ub ligase. (H and I) HEK293T cells were transfected with HA-Nedd4 or HA-Hectd1 along with Myc-Hsp90. They were then HA immunoprocipitated and subjected to Western blotting (H), or Hsp90 (I) was immunoprecipitated from E12.5 Hectd1 W and Hectd1 O embryo head lysates followed by Western blotting to detect Nedd4 and Hectd1. All data are representative of three independent experiments unless otherwise indicated. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation.

    Journal: The Journal of Cell Biology

    Article Title: Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

    doi: 10.1083/jcb.201105101

    Figure Lengend Snippet: Hectd1 physically interacts with Hsp90α. (A) Yeast two-hybrid screening of an E11.5 embryonic mouse cDNA library using Hectd1 ANK as bait detected Hsp90α (Gene ID: Hsp90aa1). Two identical clones of Hsp90α consisting of amino acids 241–459 (Hsp90αbd) of the 732–amino acid Hsp90α protein partially overlap with the first charged domain (CD1, amino acids 236–272) and the ATPase domain (amino acids 272–618) of Hsp90α. Black dots indicate locations of Hsp90 peptide fragments found by LC-MS analysis: NPDDITQEEYGEFYK (300–315), TLTIVDTGIGMTK (88–100), KADLINNLGTIAKS (100–113), and GVVDSEDLPLNISR (387–400). Peptides common to Hsp90β include: KEDQTEYLEERR (190–201), RDNSTMGYMMAKK (620–632), and YIDQEELNK (284–292). The C-terminal amino acid sequence MEEVD of Hsp90α is essential for regulated secretion. (B and C) Liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomic screening of Hectd1-binding proteins from E10.5 Hectd1 W embryo head lysates. Hectd1 was immunoprecipitated and associated proteins were resolved by 3–8% Tris-Acetate SDS-PAGE and visualized by Coomassie staining. (B) Individual bands from the Coomassie-stained gel were subjected to tryptic proteolysis, and the resulting peptides were analyzed by LC-MS/MS. (C) Representative MS/MS of a 1,293.54 D peptide. This and six other peptides (dots in A) with high XC scores (3.3–3.7) were identified as belonging to Hsp90α and Hsp90β when searched against the mouse Uniprot protein database using the Sequest algorithm as diagrammed in A. (D) Hsp90αbd binds to Hectd1 ANK in rabbit reticulocyte lysates. In vitro translated, biotinylated Hsp90αbd and Hectd1 ANK were bound and immunoprecipitated using the indicated antibodies and detected by Western blotting with streptavidin-HRP. (E and F) Hectd1 binds to Hsp90 in HEK293T cells. Cells were transfected and immunoprecipitated proteins were subjected to Western blot analyses as indicated. (E) Hsp90αbd binds to Hectd1 ANK in HEK293T cells. (F) Full-length Hsp90 and Hectd1 bind in HEK293T cells. (G) Hsp90 binds to Hectd1 in the developing embryo. Hectd1 was immunoprecipitated from lysates prepared from E12.5 Hectd1 W and Hectd1 O embryo heads and subjected to Western blot analysis as indicated ( n = 4). (H and I) Hsp90 binds to Hectd1 but not the related HECT domain containing Nedd4 Ub ligase. (H and I) HEK293T cells were transfected with HA-Nedd4 or HA-Hectd1 along with Myc-Hsp90. They were then HA immunoprocipitated and subjected to Western blotting (H), or Hsp90 (I) was immunoprecipitated from E12.5 Hectd1 W and Hectd1 O embryo head lysates followed by Western blotting to detect Nedd4 and Hectd1. All data are representative of three independent experiments unless otherwise indicated. W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation.

    Article Snippet: For lenient binding conditions, a mild immunoprecipitation buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 with protease inhibitor cocktail) was used, and for stringent binding conditions, radioimmunoprecipitation assay buffer (no. 89901; Thermo Fisher Scientific) with protease inhibition cocktail (Roche) was used.

    Techniques: Two Hybrid Screening, cDNA Library Assay, Clone Assay, Liquid Chromatography with Mass Spectroscopy, Sequencing, Liquid Chromatography, Mass Spectrometry, Binding Assay, Immunoprecipitation, SDS Page, Staining, In Vitro, Western Blot, Transfection

    Hectd1 is required for K63-linked Ub n of Hsp90. (A–D) HEK293T cells were transfected, and lysates were subjected to immunoprecipitation and Western blot analysis as indicated. (A) Ubiquitination of Myc-Hsp90 increases with expression of HA-Hectd1 ( n = 2). (B) siRNA-mediated knockdown of endogenous Hectd1 reduces the accumulation of HMW-Hsp90α ( n = 2). (C) Hsp90α ubiquitination utilizes K63 linkages. (D) Hectd1-dependent polyubiquitination of Hsp90 occurs primarily through K63 linkages. (E) HMW Hsp90 species are reduced in Hectd1 mutant heads. E12.5 Hectd1 W (W) and Hectd1 X (X) embryos were cultured in the presence of 10 µM MG132 for 3 h before lysis and immunoprecipitation of Hectd1. Immunoprecipitates were subjected to Western blot analyses to detect Hsp90 that coimmunoprecipitated with Hectd1. (F) Hsp90 ubiquitination is reduced in CM cultures from Hectd1 O (O) and Hectd1 X (X) mutants compared with Hectd1 W (W). Hsp90 was immunoprecipitated from E12.5 CM primary cultures in highly denaturing ubiquitination buffer plus 5% SDS and subjected to Western blot analyses as indicated. The appearance of a 30-kD ubiquitinated protein (asterisk) is reduced in Hectd1 mutant cells. All data are representative of three independent experiments unless otherwise indicated. Abbreviations: W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation; (Ub)n, Ub n ; wt-Ub, wild-type Ub; K48R, mutant Ub lysine 48 arginine; K63R, mutant Ub lysine 48 arginine; K0, lysineless Ub.

    Journal: The Journal of Cell Biology

    Article Title: Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme

    doi: 10.1083/jcb.201105101

    Figure Lengend Snippet: Hectd1 is required for K63-linked Ub n of Hsp90. (A–D) HEK293T cells were transfected, and lysates were subjected to immunoprecipitation and Western blot analysis as indicated. (A) Ubiquitination of Myc-Hsp90 increases with expression of HA-Hectd1 ( n = 2). (B) siRNA-mediated knockdown of endogenous Hectd1 reduces the accumulation of HMW-Hsp90α ( n = 2). (C) Hsp90α ubiquitination utilizes K63 linkages. (D) Hectd1-dependent polyubiquitination of Hsp90 occurs primarily through K63 linkages. (E) HMW Hsp90 species are reduced in Hectd1 mutant heads. E12.5 Hectd1 W (W) and Hectd1 X (X) embryos were cultured in the presence of 10 µM MG132 for 3 h before lysis and immunoprecipitation of Hectd1. Immunoprecipitates were subjected to Western blot analyses to detect Hsp90 that coimmunoprecipitated with Hectd1. (F) Hsp90 ubiquitination is reduced in CM cultures from Hectd1 O (O) and Hectd1 X (X) mutants compared with Hectd1 W (W). Hsp90 was immunoprecipitated from E12.5 CM primary cultures in highly denaturing ubiquitination buffer plus 5% SDS and subjected to Western blot analyses as indicated. The appearance of a 30-kD ubiquitinated protein (asterisk) is reduced in Hectd1 mutant cells. All data are representative of three independent experiments unless otherwise indicated. Abbreviations: W, Hectd1 +/+ ; O, Hectd1 opm/opm ; X, Hectd1 X/X ; WCL, whole cell lysate; IB, immunoblot; IP, immunoprecipitation; (Ub)n, Ub n ; wt-Ub, wild-type Ub; K48R, mutant Ub lysine 48 arginine; K63R, mutant Ub lysine 48 arginine; K0, lysineless Ub.

    Article Snippet: For lenient binding conditions, a mild immunoprecipitation buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 with protease inhibitor cocktail) was used, and for stringent binding conditions, radioimmunoprecipitation assay buffer (no. 89901; Thermo Fisher Scientific) with protease inhibition cocktail (Roche) was used.

    Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Mutagenesis, Cell Culture, Lysis

    Scheme of BMSC isolation. BM-PBS aspirate was divided into four fractions for comparative isolation of BMSCs: 1) untreated whole BM aspirate, 2) 3 volumes of RBC lysis with ammonium chloride, 3) 6 volumes of RBC lysis, or 4) Ficoll density-gradient centrifugation. Finally, BM-PBS mixtures were added to the cell culture medium.

    Journal: PLoS ONE

    Article Title: Comparisons of Rabbit Bone Marrow Mesenchymal Stem Cell Isolation and Culture Methods In Vitro

    doi: 10.1371/journal.pone.0088794

    Figure Lengend Snippet: Scheme of BMSC isolation. BM-PBS aspirate was divided into four fractions for comparative isolation of BMSCs: 1) untreated whole BM aspirate, 2) 3 volumes of RBC lysis with ammonium chloride, 3) 6 volumes of RBC lysis, or 4) Ficoll density-gradient centrifugation. Finally, BM-PBS mixtures were added to the cell culture medium.

    Article Snippet: 1) Isolation of BMSCs from untreated whole BM blood The BM-PBS mixture (8 ml) was centrifuged for 5 min at 1,000 rpm (Labofuge 400R, ThermoFisher, Germany) and supernatant was removed.

    Techniques: Isolation, Lysis, Gradient Centrifugation, Cell Culture