subcloning Search Results


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  • 99
    ATCC mc3t3 e1 cells
    NOTUM inhibition increases mineralization in osteoblast cell cultures. a Effect of human and mouse NOTUM conditioned media (CM) on osteoblast mineralization (alizarin red staining) in <t>MC3T3-E1</t> cells (100% human NOTUM CM = 1.2 μg·mL −1 ; 100% mouse NOTUM CM = 0.4 μg·mL −1 ) ( N = 3 cell cultures per data point). b , c Inhibition of mineralization caused by NOTUM (5% human NOTUM CM) was dose-dependently blocked by addition of NOTUM inhibitors LP-922056 or LP-914822 to the culture medium. Both studies lasted 21 days with NOTUM CM present throughout the study. Mineralizing medium = medium without addition of NOTUM CM. Values are means ± SEM of six cell cultures per data point
    Mc3t3 E1 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 649 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher subcloning efficiency dh5α competent cells
    D1 anaerobic protein purity. Protein was expressed in <t>DH5α</t> from the pASG-IBA35 vector A. Steps of D1 anaerobic purification process. Numbers above the gel mark fractions: 1 – before induction, 2- after induction, 3 – lysate, 4- pellet after centrifugation at 40 000 rcf, 5- NiNTA flow through, 6 – elution (this D1 anaerobic protein batch was used for EXAFS experiments). The marked band with mass 30 kDa corresponds to D1 protein. B. The purity of D1 anaerobic protein batch used for TSA, crystallization and SEC HPLC analysis (lanes 1 and 2).
    Subcloning Efficiency Dh5α Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 917 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene subcloning
    D1 anaerobic protein purity. Protein was expressed in <t>DH5α</t> from the pASG-IBA35 vector A. Steps of D1 anaerobic purification process. Numbers above the gel mark fractions: 1 – before induction, 2- after induction, 3 – lysate, 4- pellet after centrifugation at 40 000 rcf, 5- NiNTA flow through, 6 – elution (this D1 anaerobic protein batch was used for EXAFS experiments). The marked band with mass 30 kDa corresponds to D1 protein. B. The purity of D1 anaerobic protein batch used for TSA, crystallization and SEC HPLC analysis (lanes 1 and 2).
    Subcloning, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Addgene inc subcloning
    D1 anaerobic protein purity. Protein was expressed in <t>DH5α</t> from the pASG-IBA35 vector A. Steps of D1 anaerobic purification process. Numbers above the gel mark fractions: 1 – before induction, 2- after induction, 3 – lysate, 4- pellet after centrifugation at 40 000 rcf, 5- NiNTA flow through, 6 – elution (this D1 anaerobic protein batch was used for EXAFS experiments). The marked band with mass 30 kDa corresponds to D1 protein. B. The purity of D1 anaerobic protein batch used for TSA, crystallization and SEC HPLC analysis (lanes 1 and 2).
    Subcloning, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher topo ta cloning kit
    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged <t>PCR</t> products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a <t>TOPO-cloning</t> procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.
    Topo Ta Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 53974 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher topo shotgun subcloning kit
    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged <t>PCR</t> products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a <t>TOPO-cloning</t> procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.
    Topo Shotgun Subcloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 468 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher subcloning
    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged <t>PCR</t> products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a <t>TOPO-cloning</t> procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.
    Subcloning, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1596 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GenScript subcloning
    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged <t>PCR</t> products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a <t>TOPO-cloning</t> procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.
    Subcloning, supplied by GenScript, used in various techniques. Bioz Stars score: 94/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies subcloning
    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged <t>PCR</t> products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a <t>TOPO-cloning</t> procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.
    Subcloning, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher zero blunt topo pcr cloning kit
    Structure of pLGC09. Plasmid pLGC09 comprises three modules: a the lytic regulated module is composed of the promoter of chaperonin CCT one complex (P cct α) that drives expression of eng in P. pastoris and it is finished by aox1 transcription termination ( aox1 TT) region; b the selection marker module is composed of the translational elongation factor 1 gene promoter (P tef1 ) and of the EM7 synthetic prokaryotic promoter (P EM7 ) that drive expression of the ble gene in P. pastoris and E. coli respectively, these expressions are finished by cyc1 transcription termination region ( cyc1 TT); c the homologous recombination module is composed of the leu2 functional gene including its promoter and transcription termination region; and d the replicon <t>pCR</t> ® <t>4Blunt-TOPO</t> ® that includes the functional gene bla and pUC origin ( ori pUC )
    Zero Blunt Topo Pcr Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7046 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    OriGene subcloning
    Structure of pLGC09. Plasmid pLGC09 comprises three modules: a the lytic regulated module is composed of the promoter of chaperonin CCT one complex (P cct α) that drives expression of eng in P. pastoris and it is finished by aox1 transcription termination ( aox1 TT) region; b the selection marker module is composed of the translational elongation factor 1 gene promoter (P tef1 ) and of the EM7 synthetic prokaryotic promoter (P EM7 ) that drive expression of the ble gene in P. pastoris and E. coli respectively, these expressions are finished by cyc1 transcription termination region ( cyc1 TT); c the homologous recombination module is composed of the leu2 functional gene including its promoter and transcription termination region; and d the replicon <t>pCR</t> ® <t>4Blunt-TOPO</t> ® that includes the functional gene bla and pUC origin ( ori pUC )
    Subcloning, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC mc3t3 e1 cell line
    Effect of THSG on <t>MC3T3-E1</t> cell viability. After treatment with various concentrations of THSG (0, 5, 10, 20, 50, 80 and 100 μM) for 6, 12, 24 and 48 h, MC3T3-E1 cell viability was assessed using the MTT assay as described in Material and methods (error bar = ± SD, n = 6, * p
    Mc3t3 E1 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega subcloning
    Effect of THSG on <t>MC3T3-E1</t> cell viability. After treatment with various concentrations of THSG (0, 5, 10, 20, 50, 80 and 100 μM) for 6, 12, 24 and 48 h, MC3T3-E1 cell viability was assessed using the MTT assay as described in Material and methods (error bar = ± SD, n = 6, * p
    Subcloning, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 606 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Gene Bridges Inc bac subcloning kit
    Effect of THSG on <t>MC3T3-E1</t> cell viability. After treatment with various concentrations of THSG (0, 5, 10, 20, 50, 80 and 100 μM) for 6, 12, 24 and 48 h, MC3T3-E1 cell viability was assessed using the MTT assay as described in Material and methods (error bar = ± SD, n = 6, * p
    Bac Subcloning Kit, supplied by Gene Bridges Inc, used in various techniques. Bioz Stars score: 88/100, based on 188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC mc3t3 e1 subclone 14
    Change in F-actin organization of <t>MC3T3-E1</t> osteoblastic cells in response to no flow control ( A and B ), unidirectional steady flow ( C and D ), and oscillatory flow ( E and F ) for 1 h. Only application of steady unidirectional flow ( C and D ) resulted in
    Mc3t3 E1 Subclone 14, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    NOTUM inhibition increases mineralization in osteoblast cell cultures. a Effect of human and mouse NOTUM conditioned media (CM) on osteoblast mineralization (alizarin red staining) in MC3T3-E1 cells (100% human NOTUM CM = 1.2 μg·mL −1 ; 100% mouse NOTUM CM = 0.4 μg·mL −1 ) ( N = 3 cell cultures per data point). b , c Inhibition of mineralization caused by NOTUM (5% human NOTUM CM) was dose-dependently blocked by addition of NOTUM inhibitors LP-922056 or LP-914822 to the culture medium. Both studies lasted 21 days with NOTUM CM present throughout the study. Mineralizing medium = medium without addition of NOTUM CM. Values are means ± SEM of six cell cultures per data point

    Journal: Bone Research

    Article Title: NOTUM inhibition increases endocortical bone formation and bone strength

    doi: 10.1038/s41413-018-0038-3

    Figure Lengend Snippet: NOTUM inhibition increases mineralization in osteoblast cell cultures. a Effect of human and mouse NOTUM conditioned media (CM) on osteoblast mineralization (alizarin red staining) in MC3T3-E1 cells (100% human NOTUM CM = 1.2 μg·mL −1 ; 100% mouse NOTUM CM = 0.4 μg·mL −1 ) ( N = 3 cell cultures per data point). b , c Inhibition of mineralization caused by NOTUM (5% human NOTUM CM) was dose-dependently blocked by addition of NOTUM inhibitors LP-922056 or LP-914822 to the culture medium. Both studies lasted 21 days with NOTUM CM present throughout the study. Mineralizing medium = medium without addition of NOTUM CM. Values are means ± SEM of six cell cultures per data point

    Article Snippet: MC3T3-E1 cells (ATCC Subclone 4, catalogue # CRL-2593) were seeded at 40 000 cells/16 mm wells in growth medium (αMEM with 10% FBS) for 3 days to reach confluency.

    Techniques: Inhibition, Staining

    CREB and β-catenin cooperation promotes cAMP-dependent transcription. A. MC3T3-E1 cells were transfected with a cAMP responsive element luciferase construct. Luciferase assay was performed following stimulation of vehicle, FSK, or Wnt3a (100 ng/ml) as indicated (* P

    Journal: PLoS ONE

    Article Title: Protein Kinase A Activation Enhances β-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies

    doi: 10.1371/journal.pone.0109523

    Figure Lengend Snippet: CREB and β-catenin cooperation promotes cAMP-dependent transcription. A. MC3T3-E1 cells were transfected with a cAMP responsive element luciferase construct. Luciferase assay was performed following stimulation of vehicle, FSK, or Wnt3a (100 ng/ml) as indicated (* P

    Article Snippet: The murine pre-osteoblast cell line, MC3T3-E1, subclone 4 (CRL-2593, ATCC) was cultured in alpha modified Minimum Essential Medium (MEM α) supplemented with 10% FBS, and antibiotics.

    Techniques: Transfection, Luciferase, Construct

    Stimulation of PKA by FSK increases β-catenin phosphorylation and nuclear relocalization in MC3T3-E1 cells. A. Protein lysates were prepared from MC3T3-E1 cells treated with vehicle for Forskolin (FSK) for the times indicated and blotted with the antibodies shown. Note the increases in pS133-CREB, pS552- and pS675- β-catenin at both timepoints without changes in total protein levels. Actin is shown as a loading control. 20 µg of protein were loaded per lane. B. Immunofluorescence tracking of β-catenin after vehicle (DMSO) or FSK treatment in MC3T3-E1 cells. The bottom row shows a merged images from the panels above. Scale bar: 10 µm.

    Journal: PLoS ONE

    Article Title: Protein Kinase A Activation Enhances β-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies

    doi: 10.1371/journal.pone.0109523

    Figure Lengend Snippet: Stimulation of PKA by FSK increases β-catenin phosphorylation and nuclear relocalization in MC3T3-E1 cells. A. Protein lysates were prepared from MC3T3-E1 cells treated with vehicle for Forskolin (FSK) for the times indicated and blotted with the antibodies shown. Note the increases in pS133-CREB, pS552- and pS675- β-catenin at both timepoints without changes in total protein levels. Actin is shown as a loading control. 20 µg of protein were loaded per lane. B. Immunofluorescence tracking of β-catenin after vehicle (DMSO) or FSK treatment in MC3T3-E1 cells. The bottom row shows a merged images from the panels above. Scale bar: 10 µm.

    Article Snippet: The murine pre-osteoblast cell line, MC3T3-E1, subclone 4 (CRL-2593, ATCC) was cultured in alpha modified Minimum Essential Medium (MEM α) supplemented with 10% FBS, and antibiotics.

    Techniques: Immunofluorescence

    Mutation of phosphorylation sites affects nuclear localization of β-catenin. MC3T3-E1 cells were transfected with WT or mutated FLAG-tagged β-catenin constructs, treated with vehicle or forskolin for 6 h and then subjected to confocal microscopy with anti-FLAG antibodies. The bar graph shows the quantification of immunofluorescence data. 30-40 fields were counted for each transfection, yielding 70–100 transfected cells for each condition. Note that very little nuclear localization (

    Journal: PLoS ONE

    Article Title: Protein Kinase A Activation Enhances β-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies

    doi: 10.1371/journal.pone.0109523

    Figure Lengend Snippet: Mutation of phosphorylation sites affects nuclear localization of β-catenin. MC3T3-E1 cells were transfected with WT or mutated FLAG-tagged β-catenin constructs, treated with vehicle or forskolin for 6 h and then subjected to confocal microscopy with anti-FLAG antibodies. The bar graph shows the quantification of immunofluorescence data. 30-40 fields were counted for each transfection, yielding 70–100 transfected cells for each condition. Note that very little nuclear localization (

    Article Snippet: The murine pre-osteoblast cell line, MC3T3-E1, subclone 4 (CRL-2593, ATCC) was cultured in alpha modified Minimum Essential Medium (MEM α) supplemented with 10% FBS, and antibiotics.

    Techniques: Mutagenesis, Transfection, Construct, Confocal Microscopy, Immunofluorescence

    PKA activation promotes nuclear relocalization of phospho-β-catenin. A. Immunofluorescence for pS675-β-catenin (green), PML (red), with DAPI nuclear staining (blue) in MC3T3-E1 cells treated with vehicle (DMSO) or FSK. Note the nuclear accumulation of phospho-β-catenin in response to FSK. B. MC3T3-E1 cells were treated with vehicle or FSK and nuclear and cytosolic protein fractions prepared and blotted for the proteins shown. Specificity of the fractionation is demonstrated by blotting for Lamin A (nuclear marker) and α-tubulin (cytosolic marker). Note the enhanced phospho-β-catenin only in the nuclear fraction in response to FSK. 8 µg of nuclear and 20 µg of cytosolic protein were loaded per lane. C. Control or Prkar1a-knockdown MC3T3-E1 cells were studied by IF as in panel A. Scale bar for all images: 10 µm.

    Journal: PLoS ONE

    Article Title: Protein Kinase A Activation Enhances β-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies

    doi: 10.1371/journal.pone.0109523

    Figure Lengend Snippet: PKA activation promotes nuclear relocalization of phospho-β-catenin. A. Immunofluorescence for pS675-β-catenin (green), PML (red), with DAPI nuclear staining (blue) in MC3T3-E1 cells treated with vehicle (DMSO) or FSK. Note the nuclear accumulation of phospho-β-catenin in response to FSK. B. MC3T3-E1 cells were treated with vehicle or FSK and nuclear and cytosolic protein fractions prepared and blotted for the proteins shown. Specificity of the fractionation is demonstrated by blotting for Lamin A (nuclear marker) and α-tubulin (cytosolic marker). Note the enhanced phospho-β-catenin only in the nuclear fraction in response to FSK. 8 µg of nuclear and 20 µg of cytosolic protein were loaded per lane. C. Control or Prkar1a-knockdown MC3T3-E1 cells were studied by IF as in panel A. Scale bar for all images: 10 µm.

    Article Snippet: The murine pre-osteoblast cell line, MC3T3-E1, subclone 4 (CRL-2593, ATCC) was cultured in alpha modified Minimum Essential Medium (MEM α) supplemented with 10% FBS, and antibiotics.

    Techniques: Activation Assay, Immunofluorescence, Staining, Fractionation, Marker

    PKA activation represses Wnt5a/Ror2 pathway. A. and B. mRNA expression of Wnt5a and Ror2 was determined using QPCR analysis in MC3T3-E1 cells treated with FSK (A) or with Prkar1a knockdown (B) (** P

    Journal: PLoS ONE

    Article Title: Protein Kinase A Activation Enhances β-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies

    doi: 10.1371/journal.pone.0109523

    Figure Lengend Snippet: PKA activation represses Wnt5a/Ror2 pathway. A. and B. mRNA expression of Wnt5a and Ror2 was determined using QPCR analysis in MC3T3-E1 cells treated with FSK (A) or with Prkar1a knockdown (B) (** P

    Article Snippet: The murine pre-osteoblast cell line, MC3T3-E1, subclone 4 (CRL-2593, ATCC) was cultured in alpha modified Minimum Essential Medium (MEM α) supplemented with 10% FBS, and antibiotics.

    Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction

    PKA activation enhances basal and stimulated Wnt/β-catenin -dependent transcriptional activity in MC3T3-E1 cells. A. Cells were transfected with a Wnt/β-catenin -reporter plasmid (TOPFlash) or the same plasmid with a mutation in the Wnt-responsive elements (FOPFlash). Luciferase assay was performed following treatment with vehicle, FSK, or Wnt3a (100 ng/ml) as indicated (* P

    Journal: PLoS ONE

    Article Title: Protein Kinase A Activation Enhances β-Catenin Transcriptional Activity through Nuclear Localization to PML Bodies

    doi: 10.1371/journal.pone.0109523

    Figure Lengend Snippet: PKA activation enhances basal and stimulated Wnt/β-catenin -dependent transcriptional activity in MC3T3-E1 cells. A. Cells were transfected with a Wnt/β-catenin -reporter plasmid (TOPFlash) or the same plasmid with a mutation in the Wnt-responsive elements (FOPFlash). Luciferase assay was performed following treatment with vehicle, FSK, or Wnt3a (100 ng/ml) as indicated (* P

    Article Snippet: The murine pre-osteoblast cell line, MC3T3-E1, subclone 4 (CRL-2593, ATCC) was cultured in alpha modified Minimum Essential Medium (MEM α) supplemented with 10% FBS, and antibiotics.

    Techniques: Activation Assay, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Luciferase

    Wnt3a transcriptionally up-regulates lysyl oxidase mRNA levels in C3H10T1/2 pluripotent progenitor cells. Serum-depleted cells were treated with Wnt3a- or control-conditioned medium for 24 hours. Total RNA and protein were extracted and subjected to real time PCR and Western blotting analyses. A) The bar graph presents lysyl oxidase mRNA levels in response to Wnt3a in C3H10T1/2 cells (n = 6), mouse primary bone marrow stromal cells (BMSCs, n = 3), rat primary calvarial osteoblasts (n = 9), and a mouse pre-osteoblast cells (MC3T3-E1, n = 3). Data presented for C3H10T1/2 cells, BMSCs and primary calvarial osteoblasts were pooled from two independent experiments; data from MC3T3-E1 cells were from one experiment performed in triplicate. Data shown are means ± SD (*, p

    Journal: PLoS ONE

    Article Title: A Novel Function for Lysyl Oxidase in Pluripotent Mesenchymal Cell Proliferation and Relevance to Inflammation-Associated Osteopenia

    doi: 10.1371/journal.pone.0100669

    Figure Lengend Snippet: Wnt3a transcriptionally up-regulates lysyl oxidase mRNA levels in C3H10T1/2 pluripotent progenitor cells. Serum-depleted cells were treated with Wnt3a- or control-conditioned medium for 24 hours. Total RNA and protein were extracted and subjected to real time PCR and Western blotting analyses. A) The bar graph presents lysyl oxidase mRNA levels in response to Wnt3a in C3H10T1/2 cells (n = 6), mouse primary bone marrow stromal cells (BMSCs, n = 3), rat primary calvarial osteoblasts (n = 9), and a mouse pre-osteoblast cells (MC3T3-E1, n = 3). Data presented for C3H10T1/2 cells, BMSCs and primary calvarial osteoblasts were pooled from two independent experiments; data from MC3T3-E1 cells were from one experiment performed in triplicate. Data shown are means ± SD (*, p

    Article Snippet: Cell culture MC3T3-E1 cells (cat#CRL-2593) and C3H10T1/2 cells (cat#CCL-226) were purchased from ATCC.

    Techniques: Real-time Polymerase Chain Reaction, Western Blot

    D1 anaerobic protein purity. Protein was expressed in DH5α from the pASG-IBA35 vector A. Steps of D1 anaerobic purification process. Numbers above the gel mark fractions: 1 – before induction, 2- after induction, 3 – lysate, 4- pellet after centrifugation at 40 000 rcf, 5- NiNTA flow through, 6 – elution (this D1 anaerobic protein batch was used for EXAFS experiments). The marked band with mass 30 kDa corresponds to D1 protein. B. The purity of D1 anaerobic protein batch used for TSA, crystallization and SEC HPLC analysis (lanes 1 and 2).

    Journal: Protein expression and purification

    Article Title: Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization

    doi: 10.1016/j.pep.2017.03.019

    Figure Lengend Snippet: D1 anaerobic protein purity. Protein was expressed in DH5α from the pASG-IBA35 vector A. Steps of D1 anaerobic purification process. Numbers above the gel mark fractions: 1 – before induction, 2- after induction, 3 – lysate, 4- pellet after centrifugation at 40 000 rcf, 5- NiNTA flow through, 6 – elution (this D1 anaerobic protein batch was used for EXAFS experiments). The marked band with mass 30 kDa corresponds to D1 protein. B. The purity of D1 anaerobic protein batch used for TSA, crystallization and SEC HPLC analysis (lanes 1 and 2).

    Article Snippet: During the course of the study two E. coli strains, DH5α (#18265017, LifeTechnologies) and BL21(DE3)RILP (#230280, Agilent Technologies) were used.

    Techniques: Plasmid Preparation, Purification, Centrifugation, Flow Cytometry, Crystallization Assay, Size-exclusion Chromatography, High Performance Liquid Chromatography

    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged PCR products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a TOPO-cloning procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.

    Journal: Disease Models & Mechanisms

    Article Title: A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

    doi: 10.1242/dmm.026765

    Figure Lengend Snippet: Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged PCR products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a TOPO-cloning procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.

    Article Snippet: The resulting PCRs from the same group of fish were checked on the gel, mixed, purified using QIAquick PCR purification kit (QIAGEN, 28104) and TOPO-cloned into pCR2.1-TOPO (Thermo Fisher Scientific, 450641).

    Techniques: Mutagenesis, Sequencing, Fluorescence In Situ Hybridization, Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    Structure of pLGC09. Plasmid pLGC09 comprises three modules: a the lytic regulated module is composed of the promoter of chaperonin CCT one complex (P cct α) that drives expression of eng in P. pastoris and it is finished by aox1 transcription termination ( aox1 TT) region; b the selection marker module is composed of the translational elongation factor 1 gene promoter (P tef1 ) and of the EM7 synthetic prokaryotic promoter (P EM7 ) that drive expression of the ble gene in P. pastoris and E. coli respectively, these expressions are finished by cyc1 transcription termination region ( cyc1 TT); c the homologous recombination module is composed of the leu2 functional gene including its promoter and transcription termination region; and d the replicon pCR ® 4Blunt-TOPO ® that includes the functional gene bla and pUC origin ( ori pUC )

    Journal: AMB Express

    Article Title: Autolysis of Pichia pastoris induced by cold

    doi: 10.1186/s13568-017-0397-y

    Figure Lengend Snippet: Structure of pLGC09. Plasmid pLGC09 comprises three modules: a the lytic regulated module is composed of the promoter of chaperonin CCT one complex (P cct α) that drives expression of eng in P. pastoris and it is finished by aox1 transcription termination ( aox1 TT) region; b the selection marker module is composed of the translational elongation factor 1 gene promoter (P tef1 ) and of the EM7 synthetic prokaryotic promoter (P EM7 ) that drive expression of the ble gene in P. pastoris and E. coli respectively, these expressions are finished by cyc1 transcription termination region ( cyc1 TT); c the homologous recombination module is composed of the leu2 functional gene including its promoter and transcription termination region; and d the replicon pCR ® 4Blunt-TOPO ® that includes the functional gene bla and pUC origin ( ori pUC )

    Article Snippet: The two-modules amplicon was cloned in pCR® 4Blunt-TOPO® using the Zero Blunt® TOPO® PCR Cloning Kit for Sequencing (Invitrogen) following the instructions of the supplier.

    Techniques: Plasmid Preparation, Expressing, Selection, Marker, Homologous Recombination, Functional Assay, Polymerase Chain Reaction

    Methylation and expression analyses of K19. a Schematic representation of the location of discrete KRT19 gene promoter regions and the results of KRT19 methylation assessment by a highly sensitive fluorescence assay for bisulfite DNA (Hi-SA). The gray squares denote the coding exon regions of the KRT19 gene. The blue and green squares represent the restriction sites for Hha I; vertical lines indicate CpG sites; thick horizontal blue and green lines represent polymerase chain reaction fragments; arrows represent primers; and black arrows point out methylated alleles cleaved by the restriction enzyme. b Cloning and sequencing of KRT19 regions 1 and 2. Polymerase chain reaction products that were amplified by primer sets for bisulfite cloning were cloned into a TOPO cloning vector and sequenced. For the two cell lines, at least 12 clones were sequenced. Empty circles indicate unmethylated CpG sites. Filled circles represent methylated CpG sites. c Expression of K19 protein in cell lines and methylation rates in the KRT19 gene and LINE-1 . d Expression ratio (log 2 ratio) of KRT19 messenger RNA in the 5 cell lines (HepG2, HuH7, HLE, HLF, and PLC/PRF/5). Expression ratio denotes log 2 ratio obtained from the signal intensity of KRT19 messenger RNA from the cells treated with 5-aza-dC and TSA divided by the signal intensity of KRT19 messenger RNA from the cells untreated by a microarray analysis.

    Journal: Liver Cancer

    Article Title: Heterogeneity of Epigenetic and Epithelial Mesenchymal Transition Marks in Hepatocellular Carcinoma with Keratin 19 Proficiency

    doi: 10.1159/000490806

    Figure Lengend Snippet: Methylation and expression analyses of K19. a Schematic representation of the location of discrete KRT19 gene promoter regions and the results of KRT19 methylation assessment by a highly sensitive fluorescence assay for bisulfite DNA (Hi-SA). The gray squares denote the coding exon regions of the KRT19 gene. The blue and green squares represent the restriction sites for Hha I; vertical lines indicate CpG sites; thick horizontal blue and green lines represent polymerase chain reaction fragments; arrows represent primers; and black arrows point out methylated alleles cleaved by the restriction enzyme. b Cloning and sequencing of KRT19 regions 1 and 2. Polymerase chain reaction products that were amplified by primer sets for bisulfite cloning were cloned into a TOPO cloning vector and sequenced. For the two cell lines, at least 12 clones were sequenced. Empty circles indicate unmethylated CpG sites. Filled circles represent methylated CpG sites. c Expression of K19 protein in cell lines and methylation rates in the KRT19 gene and LINE-1 . d Expression ratio (log 2 ratio) of KRT19 messenger RNA in the 5 cell lines (HepG2, HuH7, HLE, HLF, and PLC/PRF/5). Expression ratio denotes log 2 ratio obtained from the signal intensity of KRT19 messenger RNA from the cells treated with 5-aza-dC and TSA divided by the signal intensity of KRT19 messenger RNA from the cells untreated by a microarray analysis.

    Article Snippet: Bisulfite Sequencing Polymerase chain reaction products from the KRT19 ) for bisulfite DNA cloning and sequencing.

    Techniques: Methylation, Expressing, Fluorescence, Polymerase Chain Reaction, Clone Assay, Sequencing, Amplification, Plasmid Preparation, Planar Chromatography, Microarray

    Effect of THSG on MC3T3-E1 cell viability. After treatment with various concentrations of THSG (0, 5, 10, 20, 50, 80 and 100 μM) for 6, 12, 24 and 48 h, MC3T3-E1 cell viability was assessed using the MTT assay as described in Material and methods (error bar = ± SD, n = 6, * p

    Journal: Archives of Medical Science : AMS

    Article Title: Stilbene glycoside protects osteoblasts against oxidative damage via Nrf2/HO-1 and NF-κB signaling pathways

    doi: 10.5114/aoms.2018.79937

    Figure Lengend Snippet: Effect of THSG on MC3T3-E1 cell viability. After treatment with various concentrations of THSG (0, 5, 10, 20, 50, 80 and 100 μM) for 6, 12, 24 and 48 h, MC3T3-E1 cell viability was assessed using the MTT assay as described in Material and methods (error bar = ± SD, n = 6, * p

    Article Snippet: Cell culture The MC3T3-E1 cell line, similar to osteoblasts, was purchased from the American Type Culture Collection (ATCC).

    Techniques: MTT Assay

    Change in F-actin organization of MC3T3-E1 osteoblastic cells in response to no flow control ( A and B ), unidirectional steady flow ( C and D ), and oscillatory flow ( E and F ) for 1 h. Only application of steady unidirectional flow ( C and D ) resulted in

    Journal: American journal of physiology. Cell physiology

    Article Title: The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts

    doi: 10.1152/ajpcell.00352.2005

    Figure Lengend Snippet: Change in F-actin organization of MC3T3-E1 osteoblastic cells in response to no flow control ( A and B ), unidirectional steady flow ( C and D ), and oscillatory flow ( E and F ) for 1 h. Only application of steady unidirectional flow ( C and D ) resulted in

    Article Snippet: MC3T3-E1 osteoblastic cells (subclone 14; ATCC, Manassas, VA) were cultured in α-modified minimal essential medium (αMEM; Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 1% penicillin and streptomycin (Invitrogen), and maintained at 37°C and 5% CO2 in a humidified incubator.

    Techniques: Flow Cytometry