full-length coding region Search Results


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  • 99
    Thermo Fisher full length coding region
    Full Length Coding Region, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore full length coding region
    Full Length Coding Region, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene full length coding region
    Full Length Coding Region, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc full length coding region
    Full Length Coding Region, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biotechnology Information full length coding region
    Full Length Coding Region, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MDPI full length coding regions
    Full Length Coding Regions, supplied by MDPI, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher full length cdna coding regions
    Full Length Cdna Coding Regions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Janssen full length hcv ns5a coding region
    Full Length Hcv Ns5a Coding Region, supplied by Janssen, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega full length crpam coding region
    Full Length Crpam Coding Region, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega full length stat5a coding region
    DNA binding properties of wild-type (WT) and N-terminal oligomerization mutants of Stat5 in an IL-2R reconstituted system. (A) Nuclear extracts were prepared from 293T cells that were transfected with IL-2Rβ, γ c , Jak3, and expression plasmids encoding wild-type or mutant <t>Stat5a</t> and Stat5b proteins. Transfected cells were either not stimulated or induced with 2 nM IL-2 for 30 min prior to preparation of nuclear extracts. The relative expression of the Stat5 proteins used in the EMSAs depicted in panel B are shown. Ten micrograms of the IL-2-induced nuclear extracts used in panel A were run on an 8% Tris-glycine gel and Western blotted with a pan-Stat5 antibody (Ab) (Transduction Labs). (B) W37 and K70 of Stat5a and Stat5b are important for stable tetramer formation on PRRIII. EMSAs were performed with 5 μg of nuclear extracts as in panel A and the wild-type PRRIII probe.
    Full Length Stat5a Coding Region, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Horizon Discovery full length rabep2 coding region
    SDCCAG8 associates with a characteristic set of proteins at the centrosome. ( A ) The Sdccag8 interacting proteins discovered by SILAC as centrosomal components belong to 4 functional groups, i.e. centriolar satellite components, endosomal vesicle components, tRNA synthesis complex proteins, and myosin type II motors involved in ciliogenesis. ( B ) Overview of the SDCCAG8 constructs used in this study. Numbering corresponds to amino acid positions. Gray boxes designate coiled-coil domains. ( C ) SDCCAG8 interacts with <t>RABEP2,</t> ERC1, and CEP131. FLAG-tagged full-length and truncation constructs of SDCCAG8 were immunoprecipitated from extracts of HEK293 cells transfected with each construct and analyzed by Western blot. Blots were probed for FLAG, or for endogenous RABEP2, ERC1, and CEP131 respectively. Only full-length FLAG-tagged SDCCAG8, but not truncated SDCCAG8 constructs co-immunoprecipitate RABEP2, ERC1 or CEP131, except for the C-terminal SDCCAG8 fragment that weakly co-immunoprecipitated RABEP2. ( D ) Western blotting for endogenous proteins in whole cell lysates shows equal loading. ( E ) CEP131 co-immunoprecipitates FLAG-SDCCAG8. Lysates from cells transfected with FLAG-SDCCAG8 and with non-specific control siRNA or with siCEP131 were subjected to immunoprecipitation using anti-CEP131 antibodies or normal IgG. While anti-CEP131 antibodies co-immunoprecipitated FLAG-SDCCAG8 from control lysates, CEP131 knockdown abolished immunoprecipitation of FLAG-SDCCAG8, demonstrating specificity of the interaction.
    Full Length Rabep2 Coding Region, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene human wt cep250 full length coding region
    Immunodetection of endogenous <t>CEP250</t> in mouse retinal cryosections. Immunostaining of CEP250 with rod photoreceptor marker rhodopsin (A) and acetylated α-tubulin (B). CEP250 stained mainly the outer segment of photoreceptors (CEP250 is in red, Rhodopsin and acetylated-α-tubulin in green, nuclear counterstaining by DAPI in blue). Cells expressing A609V CEP250-IT6 show longer cilia . (C) Wild-type (Wt) and mutant (Mt) CEP250-IT6 (green) co-localize with acetylated α-tubulin (red) to primary cilia in serum-starved ARPE-19 cells. Immunolabelling of CEP250 and acetylated α-tubulin show longer cilia in cells transfected with the mutant A609V CEP250-IT6 compared to Wt-CEP250-IT6. (D) Cilia length quantification in Wt- and Mt- CEP250-IT6 transfected cells. Graph shows that cilia from cells expressing mutant CEP250 were one third longer than cilia from cells expressing Wt-CEP250 (n > 30). Mean and error are shown. *** indicates high statistical significance by the t-Student test, p
    Human Wt Cep250 Full Length Coding Region, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biotechnology Information full length mouse trpa1 coding region
    EVA and AITC sensitivity overlap in rat vagal afferent neurons. EVA stimulates AITC-sensitive vagal afferent neurons and <t>TRPA1-transfected</t> COS-7 cells. (A) Fluorescent photomicrographs of cultured rat vagal afferent neurons loaded with Fura-2 AM calcium indicator dye at baseline and exposed to 3 mM EVA, 300 µ M AITC, and 100 nM CAP (white arrows = EVA-, AITC-, and CAP-responsive neurons; red arrows = CAP-only responsive). (B) Representative calcium traces showing response profiles of vagal afferent neurons exposed to 3 mM EVA, 300 µ M AITC, and 100 nM CAP. (C) Histogram showing the percentage of neurons sensitive to EVA, AITC, and CAP ( N = 75 neurons). (D) Scatterplot showing the positive linear relationship between EVA and AITC calcium response ( N = 44 neurons, P
    Full Length Mouse Trpa1 Coding Region, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GenScript full length human trpc5 coding region
    <t>TRPC5</t> induces autophagy via the CaMKKβ/AMPKα/mTOR pathway in response to chemotherapy. ( A and B ) Treatment of indicated cells with ADM for 48 h increased the basal [Ca 2+ ] i and siTRPC5 blocked the ADM-induced increase in [Ca 2+ ] i . ( C ) Exposure of indicated cells to ADM for 48 h was followed by analysis of the protein levels of p-CaMKKβ, CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K and ACTB by western blot. ( D ) The effect of TRPC5 silencing on the protein levels of p-CaMKKβ, CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K and ACTB in indicated cells exposed to ADM. ( E ) The effect of 20 µmol/L BAPTA/AM on the protein levels of p-CaMKKβ, CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells exposed to ADM. ( F ) The effect of CaMKKβ silencing on the protein of CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells treated with ADM. ( G ) The effect of AMPKα silencing on the protein levels of AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells exposed to ADM. ( H and I ) The effect of BAPTA/AM, siCaMKKβ and siAMPKα on ADM induced LC3 puncta formation in indicated cells. ( J and K ) The effect of 10 µmol/L STO-609 and 5 µmol/L compound C on ADM induced LC3 puncta formation in indicated cells. ( L ) The effect of BAPTA/AM, STO-609 and compound C on accumulation of LC3-II in MCF-7/ADM cells. Values are mean ± SEM of 3 to 6 experiments. *p
    Full Length Human Trpc5 Coding Region, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher full length orfs
    Sequence characteristics of cloned <t>ORFs.</t> (a) Plot of the distribution of the 398 chromosome 22 ORFs by GC content (%) and length (bases). Closed circles are the 331 ORFs that were isolated as acceptable clones (278) or as clones with the correct ORF but currently with a problem in the sequence (53). Dotted circles are the rest of the ORFs which were not amplifiable or clonable (67). (b) Overlap of chromosome 22 ORF clones isolated here with <t>cDNA</t> collections. Analysis of GC content and length for 398 chromosome 22 ORFs, split according to whether the gene has been isolated only by the strategy described here (SANGER, red circles), only in the cDNA collections (OTHER, green triangles), in both (BOTH, black circles), or not at all (NOT, yellow triangles).
    Full Length Orfs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Addgene inc human eef2k coding region
    Sequence characteristics of cloned <t>ORFs.</t> (a) Plot of the distribution of the 398 chromosome 22 ORFs by GC content (%) and length (bases). Closed circles are the 331 ORFs that were isolated as acceptable clones (278) or as clones with the correct ORF but currently with a problem in the sequence (53). Dotted circles are the rest of the ORFs which were not amplifiable or clonable (67). (b) Overlap of chromosome 22 ORF clones isolated here with <t>cDNA</t> collections. Analysis of GC content and length for 398 chromosome 22 ORFs, split according to whether the gene has been isolated only by the strategy described here (SANGER, red circles), only in the cDNA collections (OTHER, green triangles), in both (BOTH, black circles), or not at all (NOT, yellow triangles).
    Human Eef2k Coding Region, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins full length orfs
    Sequence characteristics of cloned <t>ORFs.</t> (a) Plot of the distribution of the 398 chromosome 22 ORFs by GC content (%) and length (bases). Closed circles are the 331 ORFs that were isolated as acceptable clones (278) or as clones with the correct ORF but currently with a problem in the sequence (53). Dotted circles are the rest of the ORFs which were not amplifiable or clonable (67). (b) Overlap of chromosome 22 ORF clones isolated here with <t>cDNA</t> collections. Analysis of GC content and length for 398 chromosome 22 ORFs, split according to whether the gene has been isolated only by the strategy described here (SANGER, red circles), only in the cDNA collections (OTHER, green triangles), in both (BOTH, black circles), or not at all (NOT, yellow triangles).
    Full Length Orfs, supplied by Eurofins, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Addgene inc full length fyn open reading frame
    Sequence characteristics of cloned <t>ORFs.</t> (a) Plot of the distribution of the 398 chromosome 22 ORFs by GC content (%) and length (bases). Closed circles are the 331 ORFs that were isolated as acceptable clones (278) or as clones with the correct ORF but currently with a problem in the sequence (53). Dotted circles are the rest of the ORFs which were not amplifiable or clonable (67). (b) Overlap of chromosome 22 ORF clones isolated here with <t>cDNA</t> collections. Analysis of GC content and length for 398 chromosome 22 ORFs, split according to whether the gene has been isolated only by the strategy described here (SANGER, red circles), only in the cDNA collections (OTHER, green triangles), in both (BOTH, black circles), or not at all (NOT, yellow triangles).
    Full Length Fyn Open Reading Frame, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    DNASTAR full length orfs
    Sequence characteristics of cloned <t>ORFs.</t> (a) Plot of the distribution of the 398 chromosome 22 ORFs by GC content (%) and length (bases). Closed circles are the 331 ORFs that were isolated as acceptable clones (278) or as clones with the correct ORF but currently with a problem in the sequence (53). Dotted circles are the rest of the ORFs which were not amplifiable or clonable (67). (b) Overlap of chromosome 22 ORF clones isolated here with <t>cDNA</t> collections. Analysis of GC content and length for 398 chromosome 22 ORFs, split according to whether the gene has been isolated only by the strategy described here (SANGER, red circles), only in the cDNA collections (OTHER, green triangles), in both (BOTH, black circles), or not at all (NOT, yellow triangles).
    Full Length Orfs, supplied by DNASTAR, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare full length human
    Sequence characteristics of cloned <t>ORFs.</t> (a) Plot of the distribution of the 398 chromosome 22 ORFs by GC content (%) and length (bases). Closed circles are the 331 ORFs that were isolated as acceptable clones (278) or as clones with the correct ORF but currently with a problem in the sequence (53). Dotted circles are the rest of the ORFs which were not amplifiable or clonable (67). (b) Overlap of chromosome 22 ORF clones isolated here with <t>cDNA</t> collections. Analysis of GC content and length for 398 chromosome 22 ORFs, split according to whether the gene has been isolated only by the strategy described here (SANGER, red circles), only in the cDNA collections (OTHER, green triangles), in both (BOTH, black circles), or not at all (NOT, yellow triangles).
    Full Length Human, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher full length orfs 21
    Sequence characteristics of cloned <t>ORFs.</t> (a) Plot of the distribution of the 398 chromosome 22 ORFs by GC content (%) and length (bases). Closed circles are the 331 ORFs that were isolated as acceptable clones (278) or as clones with the correct ORF but currently with a problem in the sequence (53). Dotted circles are the rest of the ORFs which were not amplifiable or clonable (67). (b) Overlap of chromosome 22 ORF clones isolated here with <t>cDNA</t> collections. Analysis of GC content and length for 398 chromosome 22 ORFs, split according to whether the gene has been isolated only by the strategy described here (SANGER, red circles), only in the cDNA collections (OTHER, green triangles), in both (BOTH, black circles), or not at all (NOT, yellow triangles).
    Full Length Orfs 21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene full length human ptpn23 open reading frame
    Sequence characteristics of cloned <t>ORFs.</t> (a) Plot of the distribution of the 398 chromosome 22 ORFs by GC content (%) and length (bases). Closed circles are the 331 ORFs that were isolated as acceptable clones (278) or as clones with the correct ORF but currently with a problem in the sequence (53). Dotted circles are the rest of the ORFs which were not amplifiable or clonable (67). (b) Overlap of chromosome 22 ORF clones isolated here with <t>cDNA</t> collections. Analysis of GC content and length for 398 chromosome 22 ORFs, split according to whether the gene has been isolated only by the strategy described here (SANGER, red circles), only in the cDNA collections (OTHER, green triangles), in both (BOTH, black circles), or not at all (NOT, yellow triangles).
    Full Length Human Ptpn23 Open Reading Frame, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher full length cxcr2 open reading frame
    Rare and low-frequency missense variants in <t>CXCR2</t> are associated with lower white blood cell (WBC) count. The symbol legend is in the upper left corner of the figure. A vertical line represents the mean WBC count for each study. The middle of each color-coded circle (black=MHI, white=WHI, grey=SHIP) corresponds to the mean WBC count for individuals that carry the corresponding missense variants and the size of the circles is correlated with allele frequency. The three white circles in the lower left corner of the figure are provided as references and represent variants with minor allele frequency of 0.01%, 0.1% and 0.5%. For rs2228413 (p.Arg294Gln), there is a participant from MHI that is homozygote for the rare allele (star).
    Full Length Cxcr2 Open Reading Frame, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shanghai Genechem full length human ebf1
    <t>EBF1</t> over-expression reduces cell viability and cell survival in CRC cells. (A) Cell count assay was performed to determine the cell viability of HCT-116 and HT-29 cells. Cell viability was normalized to the OD value of Day 1 for each group. (B) Colony formation assay was used to determine survival of HCT-116 and HT-29 cells; survival rates were normalized to the number of colonies in the control (100%) group, * P
    Full Length Human Ebf1, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNA binding properties of wild-type (WT) and N-terminal oligomerization mutants of Stat5 in an IL-2R reconstituted system. (A) Nuclear extracts were prepared from 293T cells that were transfected with IL-2Rβ, γ c , Jak3, and expression plasmids encoding wild-type or mutant Stat5a and Stat5b proteins. Transfected cells were either not stimulated or induced with 2 nM IL-2 for 30 min prior to preparation of nuclear extracts. The relative expression of the Stat5 proteins used in the EMSAs depicted in panel B are shown. Ten micrograms of the IL-2-induced nuclear extracts used in panel A were run on an 8% Tris-glycine gel and Western blotted with a pan-Stat5 antibody (Ab) (Transduction Labs). (B) W37 and K70 of Stat5a and Stat5b are important for stable tetramer formation on PRRIII. EMSAs were performed with 5 μg of nuclear extracts as in panel A and the wild-type PRRIII probe.

    Journal: Molecular and Cellular Biology

    Article Title: The Significance of Tetramerization in Promoter Recruitment by Stat5

    doi:

    Figure Lengend Snippet: DNA binding properties of wild-type (WT) and N-terminal oligomerization mutants of Stat5 in an IL-2R reconstituted system. (A) Nuclear extracts were prepared from 293T cells that were transfected with IL-2Rβ, γ c , Jak3, and expression plasmids encoding wild-type or mutant Stat5a and Stat5b proteins. Transfected cells were either not stimulated or induced with 2 nM IL-2 for 30 min prior to preparation of nuclear extracts. The relative expression of the Stat5 proteins used in the EMSAs depicted in panel B are shown. Ten micrograms of the IL-2-induced nuclear extracts used in panel A were run on an 8% Tris-glycine gel and Western blotted with a pan-Stat5 antibody (Ab) (Transduction Labs). (B) W37 and K70 of Stat5a and Stat5b are important for stable tetramer formation on PRRIII. EMSAs were performed with 5 μg of nuclear extracts as in panel A and the wild-type PRRIII probe.

    Article Snippet: To construct the expression plasmids pCI-Stat5a and pCI-Stat5b, a Sal I fragment containing the full-length Stat5a-coding region was cloned in the Sal I site of pCI (Promega) and a Sma I fragment containing the full-length Stat5b cDNA from pSX-Stat5b ( ) was cloned in the Sma I site of pCI.

    Techniques: Binding Assay, Transfection, Expressing, Mutagenesis, Western Blot, Transduction

    Transactivation properties of the W37 and K70 mutant forms of Stat5a and Stat5b. (A) Mutations of W37 alone and of both W37 and K70 in Stat5a and Stat5b significantly reduce their transactivation functions. 293T cells were transfected with IL-2Rβ, γ c , Jak3, and wild-type or mutant Stat5a and Stat5b proteins as well as with 1 μg of a PRRIII-luciferase reporter construct and 0.5 ng of a control luciferase plasmid, pRL-TKLuciferase. Twenty-four hours after transfection, cells were split into two groups, and one-half was stimulated with 2 nM IL-2 for 11 to 14 h. The relative luciferase activity is the activity of the reporter plasmid after normalization for the activity of the control reporter plasmid. (B) Experiments were performed as for panel A, but both Stat5a and Stat5b expression plasmids were simultaneously expressed. WT, wild type.

    Journal: Molecular and Cellular Biology

    Article Title: The Significance of Tetramerization in Promoter Recruitment by Stat5

    doi:

    Figure Lengend Snippet: Transactivation properties of the W37 and K70 mutant forms of Stat5a and Stat5b. (A) Mutations of W37 alone and of both W37 and K70 in Stat5a and Stat5b significantly reduce their transactivation functions. 293T cells were transfected with IL-2Rβ, γ c , Jak3, and wild-type or mutant Stat5a and Stat5b proteins as well as with 1 μg of a PRRIII-luciferase reporter construct and 0.5 ng of a control luciferase plasmid, pRL-TKLuciferase. Twenty-four hours after transfection, cells were split into two groups, and one-half was stimulated with 2 nM IL-2 for 11 to 14 h. The relative luciferase activity is the activity of the reporter plasmid after normalization for the activity of the control reporter plasmid. (B) Experiments were performed as for panel A, but both Stat5a and Stat5b expression plasmids were simultaneously expressed. WT, wild type.

    Article Snippet: To construct the expression plasmids pCI-Stat5a and pCI-Stat5b, a Sal I fragment containing the full-length Stat5a-coding region was cloned in the Sal I site of pCI (Promega) and a Sma I fragment containing the full-length Stat5b cDNA from pSX-Stat5b ( ) was cloned in the Sma I site of pCI.

    Techniques: Mutagenesis, Transfection, Luciferase, Construct, Plasmid Preparation, Activity Assay, Expressing

    A mutant PRRIII that contains tandem strong GAS motifs can be activated by a tetramerization-defective mutant of Stat5. (A) The sequences of wild-type (WT) PRRIII and mutants M9 and M10 are shown. EBS, Ets binding site. (B) The Stat5 W37A protein exhibits diminished activation of the M9-luciferase reporter construct. 293T cells were reconstituted as described before, except that 1 μg of the mutant reporter M9-luciferase was transfected in these experiments. (C) Stat5a and Stat5b can bind to the mutant M10 PRRIII probe equally well as dimers and 2× dimers. EMSAs were performed with 5 or 10 ng of rStat5a (lanes 1 to 4) or rStat5b (lanes 5 to 8) proteins and wild-type (lanes 1, 2, 5, and 6) or M10 (lanes 3, 4, 7, and 8) probes. The greater-mobility (dimeric) Stat5-PRRIII complex is stably formed with the M10 probe but not with the wild-type probe. The positions of the dimer and 2× dimer Stat5 complexes are indicated. (D) rStat5a W37A protein can also bind as dimers or 2× dimers to the high-affinity M10 probe. EMSAs were performed with a labeled M10 probe and approximately 40 ng of rStat5a or rStat5a W37A proteins. (E) The Stat5 W37A proteins can activate transcription of an M10-luciferase reporter construct with similar potency to that of WT Stat5 proteins. 293T cells were reconstituted as described before, except that 1 μg of the mutant reporter, M10-luciferase, was transfected in these experiments.

    Journal: Molecular and Cellular Biology

    Article Title: The Significance of Tetramerization in Promoter Recruitment by Stat5

    doi:

    Figure Lengend Snippet: A mutant PRRIII that contains tandem strong GAS motifs can be activated by a tetramerization-defective mutant of Stat5. (A) The sequences of wild-type (WT) PRRIII and mutants M9 and M10 are shown. EBS, Ets binding site. (B) The Stat5 W37A protein exhibits diminished activation of the M9-luciferase reporter construct. 293T cells were reconstituted as described before, except that 1 μg of the mutant reporter M9-luciferase was transfected in these experiments. (C) Stat5a and Stat5b can bind to the mutant M10 PRRIII probe equally well as dimers and 2× dimers. EMSAs were performed with 5 or 10 ng of rStat5a (lanes 1 to 4) or rStat5b (lanes 5 to 8) proteins and wild-type (lanes 1, 2, 5, and 6) or M10 (lanes 3, 4, 7, and 8) probes. The greater-mobility (dimeric) Stat5-PRRIII complex is stably formed with the M10 probe but not with the wild-type probe. The positions of the dimer and 2× dimer Stat5 complexes are indicated. (D) rStat5a W37A protein can also bind as dimers or 2× dimers to the high-affinity M10 probe. EMSAs were performed with a labeled M10 probe and approximately 40 ng of rStat5a or rStat5a W37A proteins. (E) The Stat5 W37A proteins can activate transcription of an M10-luciferase reporter construct with similar potency to that of WT Stat5 proteins. 293T cells were reconstituted as described before, except that 1 μg of the mutant reporter, M10-luciferase, was transfected in these experiments.

    Article Snippet: To construct the expression plasmids pCI-Stat5a and pCI-Stat5b, a Sal I fragment containing the full-length Stat5a-coding region was cloned in the Sal I site of pCI (Promega) and a Sma I fragment containing the full-length Stat5b cDNA from pSX-Stat5b ( ) was cloned in the Sma I site of pCI.

    Techniques: Mutagenesis, Binding Assay, Activation Assay, Luciferase, Construct, Transfection, Stable Transfection, Labeling

    SDCCAG8 associates with a characteristic set of proteins at the centrosome. ( A ) The Sdccag8 interacting proteins discovered by SILAC as centrosomal components belong to 4 functional groups, i.e. centriolar satellite components, endosomal vesicle components, tRNA synthesis complex proteins, and myosin type II motors involved in ciliogenesis. ( B ) Overview of the SDCCAG8 constructs used in this study. Numbering corresponds to amino acid positions. Gray boxes designate coiled-coil domains. ( C ) SDCCAG8 interacts with RABEP2, ERC1, and CEP131. FLAG-tagged full-length and truncation constructs of SDCCAG8 were immunoprecipitated from extracts of HEK293 cells transfected with each construct and analyzed by Western blot. Blots were probed for FLAG, or for endogenous RABEP2, ERC1, and CEP131 respectively. Only full-length FLAG-tagged SDCCAG8, but not truncated SDCCAG8 constructs co-immunoprecipitate RABEP2, ERC1 or CEP131, except for the C-terminal SDCCAG8 fragment that weakly co-immunoprecipitated RABEP2. ( D ) Western blotting for endogenous proteins in whole cell lysates shows equal loading. ( E ) CEP131 co-immunoprecipitates FLAG-SDCCAG8. Lysates from cells transfected with FLAG-SDCCAG8 and with non-specific control siRNA or with siCEP131 were subjected to immunoprecipitation using anti-CEP131 antibodies or normal IgG. While anti-CEP131 antibodies co-immunoprecipitated FLAG-SDCCAG8 from control lysates, CEP131 knockdown abolished immunoprecipitation of FLAG-SDCCAG8, demonstrating specificity of the interaction.

    Journal: PLoS ONE

    Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

    doi: 10.1371/journal.pone.0156081

    Figure Lengend Snippet: SDCCAG8 associates with a characteristic set of proteins at the centrosome. ( A ) The Sdccag8 interacting proteins discovered by SILAC as centrosomal components belong to 4 functional groups, i.e. centriolar satellite components, endosomal vesicle components, tRNA synthesis complex proteins, and myosin type II motors involved in ciliogenesis. ( B ) Overview of the SDCCAG8 constructs used in this study. Numbering corresponds to amino acid positions. Gray boxes designate coiled-coil domains. ( C ) SDCCAG8 interacts with RABEP2, ERC1, and CEP131. FLAG-tagged full-length and truncation constructs of SDCCAG8 were immunoprecipitated from extracts of HEK293 cells transfected with each construct and analyzed by Western blot. Blots were probed for FLAG, or for endogenous RABEP2, ERC1, and CEP131 respectively. Only full-length FLAG-tagged SDCCAG8, but not truncated SDCCAG8 constructs co-immunoprecipitate RABEP2, ERC1 or CEP131, except for the C-terminal SDCCAG8 fragment that weakly co-immunoprecipitated RABEP2. ( D ) Western blotting for endogenous proteins in whole cell lysates shows equal loading. ( E ) CEP131 co-immunoprecipitates FLAG-SDCCAG8. Lysates from cells transfected with FLAG-SDCCAG8 and with non-specific control siRNA or with siCEP131 were subjected to immunoprecipitation using anti-CEP131 antibodies or normal IgG. While anti-CEP131 antibodies co-immunoprecipitated FLAG-SDCCAG8 from control lysates, CEP131 knockdown abolished immunoprecipitation of FLAG-SDCCAG8, demonstrating specificity of the interaction.

    Article Snippet: Plasmid cloning To generate GFP-RABEP2-PACT centrosomal targeting construct, full-length RABEP2 coding region (Accession:BC058900, Clone ID:5415624, Dharmacon) was cloned in the pEGFP-C1-PACT plasmid, a gift from A.Kraemer [ ].

    Techniques: Functional Assay, Construct, Immunoprecipitation, Transfection, Western Blot

    RABEP2-PACT does not rescue siSDCCAG8 ciliogenesis defect. (A,B) hTERT-RPE1 cells were transfected with non-specific siRNA (siNS) and 24 hours later transfected with EGFP-PACT (A) or EGFP-RABEP2-PACT (B) constructs. Either of the tagged proteins localized to the centrosome and were detected with anti-GFP antibody (green). Ciliogenesis (acetylated-α-tubulin, red) was not affected in either of the situations. (C,D) h TERT-RPE1 cells were transfected with siSDCCAG8 and 24 hours later transfected with EGFP-PACT (C) or EGFP-RABEP2-PACT (D) constructs. Although, the tagged proteins localized to the centrosome (green), they failed to rescue the ciliogenesis defect (acetylated α-tubulin, red) in SDCCAG8 knockdown cells. Scale bars: 4 μ m. (E) Quantitation of the percentage of ciliated cells in (A, B, C and D), demonstrates that over expression of the EGFP-PACT or EGFP-RABEP2-PACT construct does not affect normal ciliation in siNS hTERT-RPE1 cells. Moreover, EGFP-RABEP2-PACT construct fails to rescue the ciliation defect in SDCCAG8 depleted cells (**p = 0.0068). Error bars, SEM. (F) We measured the length of cilia in EGFP-PACT and EGFP-RABEP2-PACT overexpression cells to examine whether it was affected. There was no significant difference in the length of cilia between siNS cells expressing EGFP-PACT and EGFP-RABEP2-PACT (73.08 ± 4.417 μ m, n = 3, vs. 67.80 ± 2.446 μ m, n = 3, p = 0.41). Similarly, the cilia length was uncorrected by the overexpression of EGFP-PACT and EGFP-RABEP2-PACT in siSDCCAG8 cells (13.36 ± 2.194 μ m, n = 3 vs. 14.58 ± 2.083 μ m, n = 3, p = 0.72). Error bars, SEM.

    Journal: PLoS ONE

    Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

    doi: 10.1371/journal.pone.0156081

    Figure Lengend Snippet: RABEP2-PACT does not rescue siSDCCAG8 ciliogenesis defect. (A,B) hTERT-RPE1 cells were transfected with non-specific siRNA (siNS) and 24 hours later transfected with EGFP-PACT (A) or EGFP-RABEP2-PACT (B) constructs. Either of the tagged proteins localized to the centrosome and were detected with anti-GFP antibody (green). Ciliogenesis (acetylated-α-tubulin, red) was not affected in either of the situations. (C,D) h TERT-RPE1 cells were transfected with siSDCCAG8 and 24 hours later transfected with EGFP-PACT (C) or EGFP-RABEP2-PACT (D) constructs. Although, the tagged proteins localized to the centrosome (green), they failed to rescue the ciliogenesis defect (acetylated α-tubulin, red) in SDCCAG8 knockdown cells. Scale bars: 4 μ m. (E) Quantitation of the percentage of ciliated cells in (A, B, C and D), demonstrates that over expression of the EGFP-PACT or EGFP-RABEP2-PACT construct does not affect normal ciliation in siNS hTERT-RPE1 cells. Moreover, EGFP-RABEP2-PACT construct fails to rescue the ciliation defect in SDCCAG8 depleted cells (**p = 0.0068). Error bars, SEM. (F) We measured the length of cilia in EGFP-PACT and EGFP-RABEP2-PACT overexpression cells to examine whether it was affected. There was no significant difference in the length of cilia between siNS cells expressing EGFP-PACT and EGFP-RABEP2-PACT (73.08 ± 4.417 μ m, n = 3, vs. 67.80 ± 2.446 μ m, n = 3, p = 0.41). Similarly, the cilia length was uncorrected by the overexpression of EGFP-PACT and EGFP-RABEP2-PACT in siSDCCAG8 cells (13.36 ± 2.194 μ m, n = 3 vs. 14.58 ± 2.083 μ m, n = 3, p = 0.72). Error bars, SEM.

    Article Snippet: Plasmid cloning To generate GFP-RABEP2-PACT centrosomal targeting construct, full-length RABEP2 coding region (Accession:BC058900, Clone ID:5415624, Dharmacon) was cloned in the pEGFP-C1-PACT plasmid, a gift from A.Kraemer [ ].

    Techniques: Transfection, Construct, Quantitation Assay, Over Expression, Expressing

    RABEP2 siRNA knockdown abolishes cilia formation. (A) Western blot analysis of extracts from control or siRABEP2-depleted hTERT-RPE1 cells, probed with anti-RABEP2 or anti-β-actin, as a loading control. Dharmacon SMARTpool siRNAs were used to knock down endogenous RABEP2 at high efficiency. (B) RABEP2 knockdown abolishes cilia formation in hTERT-RPE1 cells. Control, RABEP2 and SDCCAG8 depleted hTERT-RPE1 cells were serum starved for 48 hours after transfection with siRNA, fixed, and immunostained for acetylated α-tubulin to label primary cilia. Percentage of ciliated cells was determined in each case, control 92.8 ± 5.2%, n = 50; siRABEP2 15 ± 8.6%, n = 50 and siSDCCAG8 31.48 ± 1.694%, n = 50. Error bars, SEM, ****p

    Journal: PLoS ONE

    Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

    doi: 10.1371/journal.pone.0156081

    Figure Lengend Snippet: RABEP2 siRNA knockdown abolishes cilia formation. (A) Western blot analysis of extracts from control or siRABEP2-depleted hTERT-RPE1 cells, probed with anti-RABEP2 or anti-β-actin, as a loading control. Dharmacon SMARTpool siRNAs were used to knock down endogenous RABEP2 at high efficiency. (B) RABEP2 knockdown abolishes cilia formation in hTERT-RPE1 cells. Control, RABEP2 and SDCCAG8 depleted hTERT-RPE1 cells were serum starved for 48 hours after transfection with siRNA, fixed, and immunostained for acetylated α-tubulin to label primary cilia. Percentage of ciliated cells was determined in each case, control 92.8 ± 5.2%, n = 50; siRABEP2 15 ± 8.6%, n = 50 and siSDCCAG8 31.48 ± 1.694%, n = 50. Error bars, SEM, ****p

    Article Snippet: Plasmid cloning To generate GFP-RABEP2-PACT centrosomal targeting construct, full-length RABEP2 coding region (Accession:BC058900, Clone ID:5415624, Dharmacon) was cloned in the pEGFP-C1-PACT plasmid, a gift from A.Kraemer [ ].

    Techniques: Western Blot, Transfection

    Proteins identified by SILAC assay as SDCCAG8 interactors localize to the basal body or centrosome. ( A ) Co-staining with the centriolar protein γ-tubulin demonstrates centrosomal localization for the identified SDCCAG8 interacting proteins RABEP2, ERC1, and CEP131 in hTERT-RPE1 cells. Note CEP131 localization also at centriolar satellites. Scale bars: 5 μm. ( B ) Co-staining with the ciliary axoneme marker poly-glutamylated tubulin (red) demonstrates ciliary and basal body localization of RABEP2, basal body localization of ERC1 and centriolar satellite localization of CEP131 in hTERT-RPE1 cells. Scale bars: 5 μm. ( C ) Quantification of the co-localization coefficients of RABEP2, ERC1 and CEP131 with γ-tubulin positive centrosomes in cells from ( A ). In each case n > 40 centrosomes; error bars, SEM. ( D ) Quantification of the co-localization coefficients of RABEP2, ERC1 and CEP131 with polyglutamine-tubulin positive centrosomes in cells from ( B ). In each case n > 40 centrosomes; error bars, SEM.

    Journal: PLoS ONE

    Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

    doi: 10.1371/journal.pone.0156081

    Figure Lengend Snippet: Proteins identified by SILAC assay as SDCCAG8 interactors localize to the basal body or centrosome. ( A ) Co-staining with the centriolar protein γ-tubulin demonstrates centrosomal localization for the identified SDCCAG8 interacting proteins RABEP2, ERC1, and CEP131 in hTERT-RPE1 cells. Note CEP131 localization also at centriolar satellites. Scale bars: 5 μm. ( B ) Co-staining with the ciliary axoneme marker poly-glutamylated tubulin (red) demonstrates ciliary and basal body localization of RABEP2, basal body localization of ERC1 and centriolar satellite localization of CEP131 in hTERT-RPE1 cells. Scale bars: 5 μm. ( C ) Quantification of the co-localization coefficients of RABEP2, ERC1 and CEP131 with γ-tubulin positive centrosomes in cells from ( A ). In each case n > 40 centrosomes; error bars, SEM. ( D ) Quantification of the co-localization coefficients of RABEP2, ERC1 and CEP131 with polyglutamine-tubulin positive centrosomes in cells from ( B ). In each case n > 40 centrosomes; error bars, SEM.

    Article Snippet: Plasmid cloning To generate GFP-RABEP2-PACT centrosomal targeting construct, full-length RABEP2 coding region (Accession:BC058900, Clone ID:5415624, Dharmacon) was cloned in the pEGFP-C1-PACT plasmid, a gift from A.Kraemer [ ].

    Techniques: Staining, Marker

    Immunodetection of endogenous CEP250 in mouse retinal cryosections. Immunostaining of CEP250 with rod photoreceptor marker rhodopsin (A) and acetylated α-tubulin (B). CEP250 stained mainly the outer segment of photoreceptors (CEP250 is in red, Rhodopsin and acetylated-α-tubulin in green, nuclear counterstaining by DAPI in blue). Cells expressing A609V CEP250-IT6 show longer cilia . (C) Wild-type (Wt) and mutant (Mt) CEP250-IT6 (green) co-localize with acetylated α-tubulin (red) to primary cilia in serum-starved ARPE-19 cells. Immunolabelling of CEP250 and acetylated α-tubulin show longer cilia in cells transfected with the mutant A609V CEP250-IT6 compared to Wt-CEP250-IT6. (D) Cilia length quantification in Wt- and Mt- CEP250-IT6 transfected cells. Graph shows that cilia from cells expressing mutant CEP250 were one third longer than cilia from cells expressing Wt-CEP250 (n > 30). Mean and error are shown. *** indicates high statistical significance by the t-Student test, p

    Journal: PLoS ONE

    Article Title: Novel Candidate Genes and a Wide Spectrum of Structural and Point Mutations Responsible for Inherited Retinal Dystrophies Revealed by Exome Sequencing

    doi: 10.1371/journal.pone.0168966

    Figure Lengend Snippet: Immunodetection of endogenous CEP250 in mouse retinal cryosections. Immunostaining of CEP250 with rod photoreceptor marker rhodopsin (A) and acetylated α-tubulin (B). CEP250 stained mainly the outer segment of photoreceptors (CEP250 is in red, Rhodopsin and acetylated-α-tubulin in green, nuclear counterstaining by DAPI in blue). Cells expressing A609V CEP250-IT6 show longer cilia . (C) Wild-type (Wt) and mutant (Mt) CEP250-IT6 (green) co-localize with acetylated α-tubulin (red) to primary cilia in serum-starved ARPE-19 cells. Immunolabelling of CEP250 and acetylated α-tubulin show longer cilia in cells transfected with the mutant A609V CEP250-IT6 compared to Wt-CEP250-IT6. (D) Cilia length quantification in Wt- and Mt- CEP250-IT6 transfected cells. Graph shows that cilia from cells expressing mutant CEP250 were one third longer than cilia from cells expressing Wt-CEP250 (n > 30). Mean and error are shown. *** indicates high statistical significance by the t-Student test, p

    Article Snippet: Expression of wt and mutant CEP250 constructs in ARPE19 cells The mammalian expression vector (pCMV6 backbone) containing the human Wt-CEP250 full-length coding region was obtained from OriGene.

    Techniques: Immunodetection, Immunostaining, Marker, Staining, Expressing, Mutagenesis, Transfection

    EVA and AITC sensitivity overlap in rat vagal afferent neurons. EVA stimulates AITC-sensitive vagal afferent neurons and TRPA1-transfected COS-7 cells. (A) Fluorescent photomicrographs of cultured rat vagal afferent neurons loaded with Fura-2 AM calcium indicator dye at baseline and exposed to 3 mM EVA, 300 µ M AITC, and 100 nM CAP (white arrows = EVA-, AITC-, and CAP-responsive neurons; red arrows = CAP-only responsive). (B) Representative calcium traces showing response profiles of vagal afferent neurons exposed to 3 mM EVA, 300 µ M AITC, and 100 nM CAP. (C) Histogram showing the percentage of neurons sensitive to EVA, AITC, and CAP ( N = 75 neurons). (D) Scatterplot showing the positive linear relationship between EVA and AITC calcium response ( N = 44 neurons, P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Ethyl Vanillin Activates TRPA1

    doi: 10.1124/jpet.116.239384

    Figure Lengend Snippet: EVA and AITC sensitivity overlap in rat vagal afferent neurons. EVA stimulates AITC-sensitive vagal afferent neurons and TRPA1-transfected COS-7 cells. (A) Fluorescent photomicrographs of cultured rat vagal afferent neurons loaded with Fura-2 AM calcium indicator dye at baseline and exposed to 3 mM EVA, 300 µ M AITC, and 100 nM CAP (white arrows = EVA-, AITC-, and CAP-responsive neurons; red arrows = CAP-only responsive). (B) Representative calcium traces showing response profiles of vagal afferent neurons exposed to 3 mM EVA, 300 µ M AITC, and 100 nM CAP. (C) Histogram showing the percentage of neurons sensitive to EVA, AITC, and CAP ( N = 75 neurons). (D) Scatterplot showing the positive linear relationship between EVA and AITC calcium response ( N = 44 neurons, P

    Article Snippet: To generate TRPA1-green fluorescent protein (GFP), the full-length mouse TRPA1 coding region (National Center for Biotechnology Information accession number ) without stop codon was polymerase chain reaction amplified from a previously generated TRPA1 expression vector using the primers below ( ) and subcloned into the KpnI and BamHI sites of pEGFP-N1 (Clontech, Mountain View, CA).

    Techniques: Transfection, Cell Culture

    Pharmacological and genetic evidence that EVA is a TRPA1 agonist. Pretreatment with the selective TRPA1 antagonist A967079 eliminates the AITC and EVA responses. (A) Representative calcium traces comparing EVA (3 mM) and AITC (300 µ M) treatment responses before and after pretreatment with 1 µ M A967079. (B) Average EVA and AITC increase in cytosolic calcium before and after pretreatment with A967079 (EVA: N = 31 neurons, P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Ethyl Vanillin Activates TRPA1

    doi: 10.1124/jpet.116.239384

    Figure Lengend Snippet: Pharmacological and genetic evidence that EVA is a TRPA1 agonist. Pretreatment with the selective TRPA1 antagonist A967079 eliminates the AITC and EVA responses. (A) Representative calcium traces comparing EVA (3 mM) and AITC (300 µ M) treatment responses before and after pretreatment with 1 µ M A967079. (B) Average EVA and AITC increase in cytosolic calcium before and after pretreatment with A967079 (EVA: N = 31 neurons, P

    Article Snippet: To generate TRPA1-green fluorescent protein (GFP), the full-length mouse TRPA1 coding region (National Center for Biotechnology Information accession number ) without stop codon was polymerase chain reaction amplified from a previously generated TRPA1 expression vector using the primers below ( ) and subcloned into the KpnI and BamHI sites of pEGFP-N1 (Clontech, Mountain View, CA).

    Techniques:

    Intracellular glutathione reduces both AITC and EVA currents. AITC is known to activate TRPA1 via intracellular oxidation; however, the extent to which this occurs for EVA is unknown. In this figure, we compare the responses between standard intracellular solutions or with glutathione (10 mM) present. (A) Representative traces showing AITC-induced currents under control (left) and with intracellular glutathione (right). (B, left panel) Average AITC-induced current density from control ( N = 9 cells) and glutathione groups ( N = 10 cells). The presence of intracellular glutathione significantly reduced the AITC-induced current ( P = 0.049, t test). (B, right panel) Mean AITC currents normalized to the average control value. (C) Representative traces showing EVA-induced currents under control (left) and with intracellular glutathione (right). (D, left panel) Average EVA-induced current density from control ( N = 9 cells) and glutathione groups ( N = 10 cells). Intracellular glutathione significantly reduced the EVA current ( P = 0.003, t test). (D, right panel) Mean EVA currents normalized to the average control value. Currents have been normalized to cell capacitance to control for differences in cell size. * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Ethyl Vanillin Activates TRPA1

    doi: 10.1124/jpet.116.239384

    Figure Lengend Snippet: Intracellular glutathione reduces both AITC and EVA currents. AITC is known to activate TRPA1 via intracellular oxidation; however, the extent to which this occurs for EVA is unknown. In this figure, we compare the responses between standard intracellular solutions or with glutathione (10 mM) present. (A) Representative traces showing AITC-induced currents under control (left) and with intracellular glutathione (right). (B, left panel) Average AITC-induced current density from control ( N = 9 cells) and glutathione groups ( N = 10 cells). The presence of intracellular glutathione significantly reduced the AITC-induced current ( P = 0.049, t test). (B, right panel) Mean AITC currents normalized to the average control value. (C) Representative traces showing EVA-induced currents under control (left) and with intracellular glutathione (right). (D, left panel) Average EVA-induced current density from control ( N = 9 cells) and glutathione groups ( N = 10 cells). Intracellular glutathione significantly reduced the EVA current ( P = 0.003, t test). (D, right panel) Mean EVA currents normalized to the average control value. Currents have been normalized to cell capacitance to control for differences in cell size. * P

    Article Snippet: To generate TRPA1-green fluorescent protein (GFP), the full-length mouse TRPA1 coding region (National Center for Biotechnology Information accession number ) without stop codon was polymerase chain reaction amplified from a previously generated TRPA1 expression vector using the primers below ( ) and subcloned into the KpnI and BamHI sites of pEGFP-N1 (Clontech, Mountain View, CA).

    Techniques:

    TRPC5 induces autophagy via the CaMKKβ/AMPKα/mTOR pathway in response to chemotherapy. ( A and B ) Treatment of indicated cells with ADM for 48 h increased the basal [Ca 2+ ] i and siTRPC5 blocked the ADM-induced increase in [Ca 2+ ] i . ( C ) Exposure of indicated cells to ADM for 48 h was followed by analysis of the protein levels of p-CaMKKβ, CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K and ACTB by western blot. ( D ) The effect of TRPC5 silencing on the protein levels of p-CaMKKβ, CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K and ACTB in indicated cells exposed to ADM. ( E ) The effect of 20 µmol/L BAPTA/AM on the protein levels of p-CaMKKβ, CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells exposed to ADM. ( F ) The effect of CaMKKβ silencing on the protein of CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells treated with ADM. ( G ) The effect of AMPKα silencing on the protein levels of AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells exposed to ADM. ( H and I ) The effect of BAPTA/AM, siCaMKKβ and siAMPKα on ADM induced LC3 puncta formation in indicated cells. ( J and K ) The effect of 10 µmol/L STO-609 and 5 µmol/L compound C on ADM induced LC3 puncta formation in indicated cells. ( L ) The effect of BAPTA/AM, STO-609 and compound C on accumulation of LC3-II in MCF-7/ADM cells. Values are mean ± SEM of 3 to 6 experiments. *p

    Journal: Scientific Reports

    Article Title: TRPC5-induced autophagy promotes drug resistance in breast carcinoma via CaMKKβ/AMPKα/mTOR pathway

    doi: 10.1038/s41598-017-03230-w

    Figure Lengend Snippet: TRPC5 induces autophagy via the CaMKKβ/AMPKα/mTOR pathway in response to chemotherapy. ( A and B ) Treatment of indicated cells with ADM for 48 h increased the basal [Ca 2+ ] i and siTRPC5 blocked the ADM-induced increase in [Ca 2+ ] i . ( C ) Exposure of indicated cells to ADM for 48 h was followed by analysis of the protein levels of p-CaMKKβ, CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K and ACTB by western blot. ( D ) The effect of TRPC5 silencing on the protein levels of p-CaMKKβ, CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K and ACTB in indicated cells exposed to ADM. ( E ) The effect of 20 µmol/L BAPTA/AM on the protein levels of p-CaMKKβ, CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells exposed to ADM. ( F ) The effect of CaMKKβ silencing on the protein of CaMKKβ, p-AMPKα, AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells treated with ADM. ( G ) The effect of AMPKα silencing on the protein levels of AMPKα, p-mTOR, mTOR, p-p70S6K, p70S6K, LC3 and ACTB in indicated cells exposed to ADM. ( H and I ) The effect of BAPTA/AM, siCaMKKβ and siAMPKα on ADM induced LC3 puncta formation in indicated cells. ( J and K ) The effect of 10 µmol/L STO-609 and 5 µmol/L compound C on ADM induced LC3 puncta formation in indicated cells. ( L ) The effect of BAPTA/AM, STO-609 and compound C on accumulation of LC3-II in MCF-7/ADM cells. Values are mean ± SEM of 3 to 6 experiments. *p

    Article Snippet: Overexpression of human TRPC5 A plasmid pcDNA3.1-TRPC5 containing the full-length human TRPC5 coding region (NM_012471.2) was from GenScript Co. Transfection with pcDNA3.1-TRPC5 plasmid was carried out using the Lipofectamine 2000 Transfection Reagent (Invitrogen) according the manufacturer’s instructions.

    Techniques: Western Blot

    Chemotherapy induced autophagy is regulated by TRPC5 in breast carcinoma cells. ( A ) MCF-7 and MDA-MB 231 cells were transfected with siTRPC5 or siCTL for 24 h and then exposed to ADM for 48 h. The levels of TRPC5, LC3, and ACTB were quantified by Western blot. ( B ) The effect of TRPC5 knockdown on the average number of LC3 dots per cell in the indicated cells. ( C ) Representative western blots and densitometric analysis normalized to ACTB demonstrating the effect of TRPC5 silencing on the accumulation of LC3-II and the average number of LC3 dots per cell in MCF-7/ADM cells. ( D and E ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of TRPC5 overexpression on accumulation of LC3-II and average number of LC3 dots per cell in indicated cells. ( F ) The effect of TRPC5 overexpression on ADM-induced LC3 puncta formation in indicated cells. Values are mean ± SEM of 3 to 6 experiments. *p

    Journal: Scientific Reports

    Article Title: TRPC5-induced autophagy promotes drug resistance in breast carcinoma via CaMKKβ/AMPKα/mTOR pathway

    doi: 10.1038/s41598-017-03230-w

    Figure Lengend Snippet: Chemotherapy induced autophagy is regulated by TRPC5 in breast carcinoma cells. ( A ) MCF-7 and MDA-MB 231 cells were transfected with siTRPC5 or siCTL for 24 h and then exposed to ADM for 48 h. The levels of TRPC5, LC3, and ACTB were quantified by Western blot. ( B ) The effect of TRPC5 knockdown on the average number of LC3 dots per cell in the indicated cells. ( C ) Representative western blots and densitometric analysis normalized to ACTB demonstrating the effect of TRPC5 silencing on the accumulation of LC3-II and the average number of LC3 dots per cell in MCF-7/ADM cells. ( D and E ) Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of TRPC5 overexpression on accumulation of LC3-II and average number of LC3 dots per cell in indicated cells. ( F ) The effect of TRPC5 overexpression on ADM-induced LC3 puncta formation in indicated cells. Values are mean ± SEM of 3 to 6 experiments. *p

    Article Snippet: Overexpression of human TRPC5 A plasmid pcDNA3.1-TRPC5 containing the full-length human TRPC5 coding region (NM_012471.2) was from GenScript Co. Transfection with pcDNA3.1-TRPC5 plasmid was carried out using the Lipofectamine 2000 Transfection Reagent (Invitrogen) according the manufacturer’s instructions.

    Techniques: Multiple Displacement Amplification, Transfection, Western Blot, Over Expression

    Signaling connections involved in TRPC5-initiated autophagy pathways in response to chemotherapy in breast cancer cells. Chemotherapy up-regulates TRPC5 expression and then increases the basal [Ca 2+ ] i . The increased [Ca 2+ ] i actives CaMKKβ by phosphorylation, which in turn activates AMPKα by phosphorylation. Activation of AMPKα negatively regulates mTOR by suppressing its phosphorylation, leading to autophagy. Autophagy induced by TRPC5 during chemotherapy promotes the survival of human breast cancer cell. Arrows represent promotion events, blunt arrows indicate suppression events.

    Journal: Scientific Reports

    Article Title: TRPC5-induced autophagy promotes drug resistance in breast carcinoma via CaMKKβ/AMPKα/mTOR pathway

    doi: 10.1038/s41598-017-03230-w

    Figure Lengend Snippet: Signaling connections involved in TRPC5-initiated autophagy pathways in response to chemotherapy in breast cancer cells. Chemotherapy up-regulates TRPC5 expression and then increases the basal [Ca 2+ ] i . The increased [Ca 2+ ] i actives CaMKKβ by phosphorylation, which in turn activates AMPKα by phosphorylation. Activation of AMPKα negatively regulates mTOR by suppressing its phosphorylation, leading to autophagy. Autophagy induced by TRPC5 during chemotherapy promotes the survival of human breast cancer cell. Arrows represent promotion events, blunt arrows indicate suppression events.

    Article Snippet: Overexpression of human TRPC5 A plasmid pcDNA3.1-TRPC5 containing the full-length human TRPC5 coding region (NM_012471.2) was from GenScript Co. Transfection with pcDNA3.1-TRPC5 plasmid was carried out using the Lipofectamine 2000 Transfection Reagent (Invitrogen) according the manufacturer’s instructions.

    Techniques: Expressing, Activation Assay

    Silencing of TRPC5 or inhibition of autophagy increases the sensitivity of breast carcinoma cells to chemotherapy ( A and B ) MTT assays. MCF-7 and MDA-MB 231 cells were transfected with siTRPC5 or siCTL for 24 h and then exposed to 400 and 800 nmol/L ADM respectively, for 48 h. ( C ) Clonogenic recovery assays. MCF-7 and MDA-MB 231 cells transfected with siTRPC5 or siCTL for 24 h and then exposed to 5 and 10 nmol/L ADM respectively. ( D and E ) The effect of TRPC5 overexpression on cell viability and clonogenic recovery in indicated cells during ADM exposure. ( F ) The effect of 50 µmol/L chloroquine (CQ) on cell viability. ( G ) The effect of 3 µmol/L CQ on clonogenic recovery in indicated cells. ( H and I ) The effect of 50 µmol/L CQ on viability and 3 µmol/L CQ on clonogenic recovery in MCF-7/ADM cells. Values are mean ± SEM of 4 to 6 experiments. *p

    Journal: Scientific Reports

    Article Title: TRPC5-induced autophagy promotes drug resistance in breast carcinoma via CaMKKβ/AMPKα/mTOR pathway

    doi: 10.1038/s41598-017-03230-w

    Figure Lengend Snippet: Silencing of TRPC5 or inhibition of autophagy increases the sensitivity of breast carcinoma cells to chemotherapy ( A and B ) MTT assays. MCF-7 and MDA-MB 231 cells were transfected with siTRPC5 or siCTL for 24 h and then exposed to 400 and 800 nmol/L ADM respectively, for 48 h. ( C ) Clonogenic recovery assays. MCF-7 and MDA-MB 231 cells transfected with siTRPC5 or siCTL for 24 h and then exposed to 5 and 10 nmol/L ADM respectively. ( D and E ) The effect of TRPC5 overexpression on cell viability and clonogenic recovery in indicated cells during ADM exposure. ( F ) The effect of 50 µmol/L chloroquine (CQ) on cell viability. ( G ) The effect of 3 µmol/L CQ on clonogenic recovery in indicated cells. ( H and I ) The effect of 50 µmol/L CQ on viability and 3 µmol/L CQ on clonogenic recovery in MCF-7/ADM cells. Values are mean ± SEM of 4 to 6 experiments. *p

    Article Snippet: Overexpression of human TRPC5 A plasmid pcDNA3.1-TRPC5 containing the full-length human TRPC5 coding region (NM_012471.2) was from GenScript Co. Transfection with pcDNA3.1-TRPC5 plasmid was carried out using the Lipofectamine 2000 Transfection Reagent (Invitrogen) according the manufacturer’s instructions.

    Techniques: Inhibition, MTT Assay, Multiple Displacement Amplification, Transfection, Over Expression

    Chemotherapy enhances autophagy and TRPC5 expression in breast carcinoma cells. ( A ) Breast cancer cells were treated with 400, 300 and 800 nmol/L ADM for 48 h, and then the expression of TRPC5, LC3 and ACTB/β-actin was analyzed by western blot. Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of ADM on LC3-II levels. ( B ) Representative immunofluorescence images showing redistribution of the autophagic marker LC3 in breast cancer cells were captured on a confocal microscope, and the average number of LC3 dots per cell. Scale bar: 20 μm. ( C ) Representative western blots and densitometric analysis normalized to ACTB demonstrating the effect of the lysosomal protease inhibitors 10 μg/mL E64d plus pepstatin A (Pep A) on ADM-induced LC3-II accumulation. ( D ) Representative western blots and densitometric analysis normalized to ACTB demonstrating the LC3-II levels in MCF-7/ADM cells and the effect of E64d and Pep A. Values are means ± SEM of 4 to 6 experiments. *p

    Journal: Scientific Reports

    Article Title: TRPC5-induced autophagy promotes drug resistance in breast carcinoma via CaMKKβ/AMPKα/mTOR pathway

    doi: 10.1038/s41598-017-03230-w

    Figure Lengend Snippet: Chemotherapy enhances autophagy and TRPC5 expression in breast carcinoma cells. ( A ) Breast cancer cells were treated with 400, 300 and 800 nmol/L ADM for 48 h, and then the expression of TRPC5, LC3 and ACTB/β-actin was analyzed by western blot. Representative western blot and densitometric analysis normalized to ACTB demonstrating the effect of ADM on LC3-II levels. ( B ) Representative immunofluorescence images showing redistribution of the autophagic marker LC3 in breast cancer cells were captured on a confocal microscope, and the average number of LC3 dots per cell. Scale bar: 20 μm. ( C ) Representative western blots and densitometric analysis normalized to ACTB demonstrating the effect of the lysosomal protease inhibitors 10 μg/mL E64d plus pepstatin A (Pep A) on ADM-induced LC3-II accumulation. ( D ) Representative western blots and densitometric analysis normalized to ACTB demonstrating the LC3-II levels in MCF-7/ADM cells and the effect of E64d and Pep A. Values are means ± SEM of 4 to 6 experiments. *p

    Article Snippet: Overexpression of human TRPC5 A plasmid pcDNA3.1-TRPC5 containing the full-length human TRPC5 coding region (NM_012471.2) was from GenScript Co. Transfection with pcDNA3.1-TRPC5 plasmid was carried out using the Lipofectamine 2000 Transfection Reagent (Invitrogen) according the manufacturer’s instructions.

    Techniques: Expressing, Western Blot, Immunofluorescence, Marker, Microscopy

    Suppression of autophagy by down-regulated TRPC5 increases sensitivity to ADM in vivo . ( A ) Female nude mice were inoculated with MCF-7 or MDA-MB 231 cells transfected with control or TRPC5 shRNA lentiviral particles and treated with ADM (6 mg/kg) when the tumors reached ~100 mm 3 (n = 5 in each group). Autophagy in tumor samples were assayed by LC3 stain. Scale bar: 100 μm. ( B and C ) Representative images and summary data from immunohistochemical staining of TRPC5 and LC3 in paired pre- and post-chemotherapy breast cancer tissue from patients showing elevated TRPC5 or LC3 expression (n = 31). Scale bar: 100 μm. ( D ) Pearson correction of TRPC5 expression with LC3 (n = 31). Data were analyzed using Pearson correlation test. Values are mean ± SEM *p

    Journal: Scientific Reports

    Article Title: TRPC5-induced autophagy promotes drug resistance in breast carcinoma via CaMKKβ/AMPKα/mTOR pathway

    doi: 10.1038/s41598-017-03230-w

    Figure Lengend Snippet: Suppression of autophagy by down-regulated TRPC5 increases sensitivity to ADM in vivo . ( A ) Female nude mice were inoculated with MCF-7 or MDA-MB 231 cells transfected with control or TRPC5 shRNA lentiviral particles and treated with ADM (6 mg/kg) when the tumors reached ~100 mm 3 (n = 5 in each group). Autophagy in tumor samples were assayed by LC3 stain. Scale bar: 100 μm. ( B and C ) Representative images and summary data from immunohistochemical staining of TRPC5 and LC3 in paired pre- and post-chemotherapy breast cancer tissue from patients showing elevated TRPC5 or LC3 expression (n = 31). Scale bar: 100 μm. ( D ) Pearson correction of TRPC5 expression with LC3 (n = 31). Data were analyzed using Pearson correlation test. Values are mean ± SEM *p

    Article Snippet: Overexpression of human TRPC5 A plasmid pcDNA3.1-TRPC5 containing the full-length human TRPC5 coding region (NM_012471.2) was from GenScript Co. Transfection with pcDNA3.1-TRPC5 plasmid was carried out using the Lipofectamine 2000 Transfection Reagent (Invitrogen) according the manufacturer’s instructions.

    Techniques: In Vivo, Mouse Assay, Multiple Displacement Amplification, Transfection, shRNA, Staining, Immunohistochemistry, Expressing

    Sequence characteristics of cloned ORFs. (a) Plot of the distribution of the 398 chromosome 22 ORFs by GC content (%) and length (bases). Closed circles are the 331 ORFs that were isolated as acceptable clones (278) or as clones with the correct ORF but currently with a problem in the sequence (53). Dotted circles are the rest of the ORFs which were not amplifiable or clonable (67). (b) Overlap of chromosome 22 ORF clones isolated here with cDNA collections. Analysis of GC content and length for 398 chromosome 22 ORFs, split according to whether the gene has been isolated only by the strategy described here (SANGER, red circles), only in the cDNA collections (OTHER, green triangles), in both (BOTH, black circles), or not at all (NOT, yellow triangles).

    Journal: Genome Biology

    Article Title: A genome annotation-driven approach to cloning the human ORFeome

    doi: 10.1186/gb-2004-5-10-r84

    Figure Lengend Snippet: Sequence characteristics of cloned ORFs. (a) Plot of the distribution of the 398 chromosome 22 ORFs by GC content (%) and length (bases). Closed circles are the 331 ORFs that were isolated as acceptable clones (278) or as clones with the correct ORF but currently with a problem in the sequence (53). Dotted circles are the rest of the ORFs which were not amplifiable or clonable (67). (b) Overlap of chromosome 22 ORF clones isolated here with cDNA collections. Analysis of GC content and length for 398 chromosome 22 ORFs, split according to whether the gene has been isolated only by the strategy described here (SANGER, red circles), only in the cDNA collections (OTHER, green triangles), in both (BOTH, black circles), or not at all (NOT, yellow triangles).

    Article Snippet: The aim of the method is to provide cDNA clones containing confirmed full-length ORFs, which can later be manipulated into suitable vector systems such as Gateway (Invitrogen) or Creator (BD Biosciences) for functional genomics.

    Techniques: Sequencing, Clone Assay, Isolation

    Schematic Venn diagram showing the relationships of the set of ORF clones isolated here compared with the full-length cDNA clones in current high-throughput clone collections (227 maintain the correct reading frame at the amino acid level from Table 1) for the 398 annotated full-length chromosome 22 ORFs. The four different classes of genes are labeled as in the text and Figure 2b.

    Journal: Genome Biology

    Article Title: A genome annotation-driven approach to cloning the human ORFeome

    doi: 10.1186/gb-2004-5-10-r84

    Figure Lengend Snippet: Schematic Venn diagram showing the relationships of the set of ORF clones isolated here compared with the full-length cDNA clones in current high-throughput clone collections (227 maintain the correct reading frame at the amino acid level from Table 1) for the 398 annotated full-length chromosome 22 ORFs. The four different classes of genes are labeled as in the text and Figure 2b.

    Article Snippet: The aim of the method is to provide cDNA clones containing confirmed full-length ORFs, which can later be manipulated into suitable vector systems such as Gateway (Invitrogen) or Creator (BD Biosciences) for functional genomics.

    Techniques: Clone Assay, Isolation, High Throughput Screening Assay, Labeling

    Rare and low-frequency missense variants in CXCR2 are associated with lower white blood cell (WBC) count. The symbol legend is in the upper left corner of the figure. A vertical line represents the mean WBC count for each study. The middle of each color-coded circle (black=MHI, white=WHI, grey=SHIP) corresponds to the mean WBC count for individuals that carry the corresponding missense variants and the size of the circles is correlated with allele frequency. The three white circles in the lower left corner of the figure are provided as references and represent variants with minor allele frequency of 0.01%, 0.1% and 0.5%. For rs2228413 (p.Arg294Gln), there is a participant from MHI that is homozygote for the rare allele (star).

    Journal: Nature genetics

    Article Title: Rare and low-frequency coding variants in CXCR2 and other genes are associated with hematological traits

    doi: 10.1038/ng.2962

    Figure Lengend Snippet: Rare and low-frequency missense variants in CXCR2 are associated with lower white blood cell (WBC) count. The symbol legend is in the upper left corner of the figure. A vertical line represents the mean WBC count for each study. The middle of each color-coded circle (black=MHI, white=WHI, grey=SHIP) corresponds to the mean WBC count for individuals that carry the corresponding missense variants and the size of the circles is correlated with allele frequency. The three white circles in the lower left corner of the figure are provided as references and represent variants with minor allele frequency of 0.01%, 0.1% and 0.5%. For rs2228413 (p.Arg294Gln), there is a participant from MHI that is homozygote for the rare allele (star).

    Article Snippet: IMAGE clone 5752441 containing the full-length CXCR2 open reading frame was obtained from Invitrogen.

    Techniques:

    Functional characterization of the CXCR2fs mutant receptor. (A) Intracellular ERK1/2 signaling in HeLa cells transfected with wild type or mutant CXCR2 receptor. HeLa cells transfected with CXCR2wt-YFP, CXCR2fs-YFP or YFP alone were stimulated with CXCL8 (100 ng/mL) and lysates collected over the time course indicated to assess the activation of the ERK1/2 pathway. Peak phosphorylation following stimulation of CXCR2wt occurred at 5 min with significant attenuation of activation by 15 min. No activation over baseline was detected in cells expressing CXCR2fs. ERK1/2 activation at 5 min following stimulation of endogenous CXCR4 with CXCL12 (100 ng/mL) was used as a positive control (+). Protein abundance of CXCR2wt-YFP (wt), CXCR2fs-YFP (fs) and the YPF control (Y) proteins in transfected cell lines are shown (right panel). (B) Chemotaxis assay results for HeLa cells expressing CXCR2wt-YFP or CXCR2fs-YFP. Transiently transfected HeLa cells were seeded in the top chamber of modified Boyden chambers and CXCL8 (100 ng/mL) was added to media in the bottom chamber. The chemotactic index (migration in presence of CXCL8/migration in absence of CXCL8) was calculated from the results of assays performed in triplicate. Robust migration was observed in cells expressing the wild type CXCR2 fusion protein (gray bars), but not in those expressing the frameshift mutant (open bars) or in untransfected cells (black bars). Overexpression of each construct was confirmed by detecting YFP fluorescence (expressed as relative fluorescence units; RFU) in aliquots of transfectants (right panel). * P

    Journal: Nature genetics

    Article Title: Rare and low-frequency coding variants in CXCR2 and other genes are associated with hematological traits

    doi: 10.1038/ng.2962

    Figure Lengend Snippet: Functional characterization of the CXCR2fs mutant receptor. (A) Intracellular ERK1/2 signaling in HeLa cells transfected with wild type or mutant CXCR2 receptor. HeLa cells transfected with CXCR2wt-YFP, CXCR2fs-YFP or YFP alone were stimulated with CXCL8 (100 ng/mL) and lysates collected over the time course indicated to assess the activation of the ERK1/2 pathway. Peak phosphorylation following stimulation of CXCR2wt occurred at 5 min with significant attenuation of activation by 15 min. No activation over baseline was detected in cells expressing CXCR2fs. ERK1/2 activation at 5 min following stimulation of endogenous CXCR4 with CXCL12 (100 ng/mL) was used as a positive control (+). Protein abundance of CXCR2wt-YFP (wt), CXCR2fs-YFP (fs) and the YPF control (Y) proteins in transfected cell lines are shown (right panel). (B) Chemotaxis assay results for HeLa cells expressing CXCR2wt-YFP or CXCR2fs-YFP. Transiently transfected HeLa cells were seeded in the top chamber of modified Boyden chambers and CXCL8 (100 ng/mL) was added to media in the bottom chamber. The chemotactic index (migration in presence of CXCL8/migration in absence of CXCL8) was calculated from the results of assays performed in triplicate. Robust migration was observed in cells expressing the wild type CXCR2 fusion protein (gray bars), but not in those expressing the frameshift mutant (open bars) or in untransfected cells (black bars). Overexpression of each construct was confirmed by detecting YFP fluorescence (expressed as relative fluorescence units; RFU) in aliquots of transfectants (right panel). * P

    Article Snippet: IMAGE clone 5752441 containing the full-length CXCR2 open reading frame was obtained from Invitrogen.

    Techniques: Functional Assay, Mutagenesis, Transfection, Activation Assay, Expressing, Positive Control, Chemotaxis Assay, Modification, Migration, Over Expression, Construct, Fluorescence

    EBF1 over-expression reduces cell viability and cell survival in CRC cells. (A) Cell count assay was performed to determine the cell viability of HCT-116 and HT-29 cells. Cell viability was normalized to the OD value of Day 1 for each group. (B) Colony formation assay was used to determine survival of HCT-116 and HT-29 cells; survival rates were normalized to the number of colonies in the control (100%) group, * P

    Journal: Frontiers in Oncology

    Article Title: Transcription Factor EBF1 Over-Expression Suppresses Tumor Growth in vivo and in vitro via Modulation of the PNO1/p53 Pathway in Colorectal Cancer

    doi: 10.3389/fonc.2020.01035

    Figure Lengend Snippet: EBF1 over-expression reduces cell viability and cell survival in CRC cells. (A) Cell count assay was performed to determine the cell viability of HCT-116 and HT-29 cells. Cell viability was normalized to the OD value of Day 1 for each group. (B) Colony formation assay was used to determine survival of HCT-116 and HT-29 cells; survival rates were normalized to the number of colonies in the control (100%) group, * P

    Article Snippet: Generation of Stable Transduction Cell Lines To generate a cell line stably over-expressing EBF1, HCT-116, or HT-29 cells were seeded in six-well plates (5 × 104 cells/well) and transduced with a lentiviral vector encoding full-length human EBF1 (coding region of 756 bp; Shanghai, GeneChem) or empty vector for 72 h. Transduced cells were further selected for 2 weeks using puromycin (Thermo Fisher Scientific) at 1 μg/ml.

    Techniques: Over Expression, Cell Counting, Colony Assay

    EBF1 over-expression down-regulates PNO1 and up-regulates p53 and p21 expression. (A,B) HCT-116 and HT-29 cells were stably transduced with EBF1 or control plasmid, and (A) Q-PCR and (B) western blotting were performed to determine the expression of PNO1, GAPDH was used as a loading control. (C) Luciferase assay was performed to determine the effect of EBF1 over-expression on PNO1 transcription in both HCT-116 and HT-29 cells. (D) Western blotting was performed to determine the protein expression of p53 and p21 in HCT-116 and HT-29 cells after EBF over-expression. GAPDH was used as a loading control. * P

    Journal: Frontiers in Oncology

    Article Title: Transcription Factor EBF1 Over-Expression Suppresses Tumor Growth in vivo and in vitro via Modulation of the PNO1/p53 Pathway in Colorectal Cancer

    doi: 10.3389/fonc.2020.01035

    Figure Lengend Snippet: EBF1 over-expression down-regulates PNO1 and up-regulates p53 and p21 expression. (A,B) HCT-116 and HT-29 cells were stably transduced with EBF1 or control plasmid, and (A) Q-PCR and (B) western blotting were performed to determine the expression of PNO1, GAPDH was used as a loading control. (C) Luciferase assay was performed to determine the effect of EBF1 over-expression on PNO1 transcription in both HCT-116 and HT-29 cells. (D) Western blotting was performed to determine the protein expression of p53 and p21 in HCT-116 and HT-29 cells after EBF over-expression. GAPDH was used as a loading control. * P

    Article Snippet: Generation of Stable Transduction Cell Lines To generate a cell line stably over-expressing EBF1, HCT-116, or HT-29 cells were seeded in six-well plates (5 × 104 cells/well) and transduced with a lentiviral vector encoding full-length human EBF1 (coding region of 756 bp; Shanghai, GeneChem) or empty vector for 72 h. Transduced cells were further selected for 2 weeks using puromycin (Thermo Fisher Scientific) at 1 μg/ml.

    Techniques: Over Expression, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Polymerase Chain Reaction, Western Blot, Luciferase

    Low EBF1 expression correlates with shorter survival in CRC patients. (A–E) A public clinical microarray dataset from the R2 bioinformatic platform was used to analyze the correlation between EBF1 mRNA expression and overall survival (A) , relapse-free survival (B) , and event-free survival (C–E) of CRC patients.

    Journal: Frontiers in Oncology

    Article Title: Transcription Factor EBF1 Over-Expression Suppresses Tumor Growth in vivo and in vitro via Modulation of the PNO1/p53 Pathway in Colorectal Cancer

    doi: 10.3389/fonc.2020.01035

    Figure Lengend Snippet: Low EBF1 expression correlates with shorter survival in CRC patients. (A–E) A public clinical microarray dataset from the R2 bioinformatic platform was used to analyze the correlation between EBF1 mRNA expression and overall survival (A) , relapse-free survival (B) , and event-free survival (C–E) of CRC patients.

    Article Snippet: Generation of Stable Transduction Cell Lines To generate a cell line stably over-expressing EBF1, HCT-116, or HT-29 cells were seeded in six-well plates (5 × 104 cells/well) and transduced with a lentiviral vector encoding full-length human EBF1 (coding region of 756 bp; Shanghai, GeneChem) or empty vector for 72 h. Transduced cells were further selected for 2 weeks using puromycin (Thermo Fisher Scientific) at 1 μg/ml.

    Techniques: Expressing, Microarray

    EBF1 over-expression suppresses CRC cell growth in vitro . (A,B) HCT-116 and HT-29 cells were stably transduced with EBF1 or control. The cells were evaluated at a fixed time in culture. (A) Q-PCR and (B) western blot were performed to determine the expression of EBF1. GAPDH was used as an internal control. (C) Cell confluence of transduced HCT-116 and HT-29 cells were imaged using a phase contrast fluorescence microscope in light at a magnification of 200×. (D) Cell number was determined with a Countstar Automated Cell Counter. * P

    Journal: Frontiers in Oncology

    Article Title: Transcription Factor EBF1 Over-Expression Suppresses Tumor Growth in vivo and in vitro via Modulation of the PNO1/p53 Pathway in Colorectal Cancer

    doi: 10.3389/fonc.2020.01035

    Figure Lengend Snippet: EBF1 over-expression suppresses CRC cell growth in vitro . (A,B) HCT-116 and HT-29 cells were stably transduced with EBF1 or control. The cells were evaluated at a fixed time in culture. (A) Q-PCR and (B) western blot were performed to determine the expression of EBF1. GAPDH was used as an internal control. (C) Cell confluence of transduced HCT-116 and HT-29 cells were imaged using a phase contrast fluorescence microscope in light at a magnification of 200×. (D) Cell number was determined with a Countstar Automated Cell Counter. * P

    Article Snippet: Generation of Stable Transduction Cell Lines To generate a cell line stably over-expressing EBF1, HCT-116, or HT-29 cells were seeded in six-well plates (5 × 104 cells/well) and transduced with a lentiviral vector encoding full-length human EBF1 (coding region of 756 bp; Shanghai, GeneChem) or empty vector for 72 h. Transduced cells were further selected for 2 weeks using puromycin (Thermo Fisher Scientific) at 1 μg/ml.

    Techniques: Over Expression, In Vitro, Stable Transfection, Transduction, Polymerase Chain Reaction, Western Blot, Expressing, Fluorescence, Microscopy

    EBF1 over-expression suppresses CRC cell growth in vivo . Stably transduced HCT-116 and HT-29 cells over expressing EBF1 or control were injected subcutaneously into BALB/C nude mice. (A) Tumor volume was measured on the indicated days and (B) tumor weight was determined at the end of experiments. * P

    Journal: Frontiers in Oncology

    Article Title: Transcription Factor EBF1 Over-Expression Suppresses Tumor Growth in vivo and in vitro via Modulation of the PNO1/p53 Pathway in Colorectal Cancer

    doi: 10.3389/fonc.2020.01035

    Figure Lengend Snippet: EBF1 over-expression suppresses CRC cell growth in vivo . Stably transduced HCT-116 and HT-29 cells over expressing EBF1 or control were injected subcutaneously into BALB/C nude mice. (A) Tumor volume was measured on the indicated days and (B) tumor weight was determined at the end of experiments. * P

    Article Snippet: Generation of Stable Transduction Cell Lines To generate a cell line stably over-expressing EBF1, HCT-116, or HT-29 cells were seeded in six-well plates (5 × 104 cells/well) and transduced with a lentiviral vector encoding full-length human EBF1 (coding region of 756 bp; Shanghai, GeneChem) or empty vector for 72 h. Transduced cells were further selected for 2 weeks using puromycin (Thermo Fisher Scientific) at 1 μg/ml.

    Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Injection, Mouse Assay

    EBF1 over-expression induces cell cycle arrest and cell apoptosis in CRC cells. (A) Propidium iodide staining and flow cytometry analysis were performed to determine cell cycle progression of HCT-116 (upper panel) and HT-29 (lower panel) cells after EBF1 over-expression. The representative images (left panel) and analysis of cell cycle distributions (right panel) are shown. (B) Annexin V staining and flow cytometry analysis were performed to measure apoptosis of HCT-116 (upper panel) and HT-29 (lower panel) cells after EBF1 over-expression. Representative images (left panel) are presented and the percentages of apoptotic cells calculated (right panel). * P

    Journal: Frontiers in Oncology

    Article Title: Transcription Factor EBF1 Over-Expression Suppresses Tumor Growth in vivo and in vitro via Modulation of the PNO1/p53 Pathway in Colorectal Cancer

    doi: 10.3389/fonc.2020.01035

    Figure Lengend Snippet: EBF1 over-expression induces cell cycle arrest and cell apoptosis in CRC cells. (A) Propidium iodide staining and flow cytometry analysis were performed to determine cell cycle progression of HCT-116 (upper panel) and HT-29 (lower panel) cells after EBF1 over-expression. The representative images (left panel) and analysis of cell cycle distributions (right panel) are shown. (B) Annexin V staining and flow cytometry analysis were performed to measure apoptosis of HCT-116 (upper panel) and HT-29 (lower panel) cells after EBF1 over-expression. Representative images (left panel) are presented and the percentages of apoptotic cells calculated (right panel). * P

    Article Snippet: Generation of Stable Transduction Cell Lines To generate a cell line stably over-expressing EBF1, HCT-116, or HT-29 cells were seeded in six-well plates (5 × 104 cells/well) and transduced with a lentiviral vector encoding full-length human EBF1 (coding region of 756 bp; Shanghai, GeneChem) or empty vector for 72 h. Transduced cells were further selected for 2 weeks using puromycin (Thermo Fisher Scientific) at 1 μg/ml.

    Techniques: Over Expression, Staining, Flow Cytometry

    EBF1 is down-regulated in human colorectal cancer. (A) EBF1 mRNA expression (probe1: 11726800_at; probe2: 11726801_at; probe3: 11726802_a_at) in 14 matched CRC and non-cancerous colorectal tissues from our previously described gene expression profile microarrays (GEO Submission: GSE113513). Each dot represents 1 tissue. (B,C) EBF1 mRNA expression in (B) COAD tissues ( n = 279) and normal colon tissues ( n = 345), (C) READ tissues ( n = 92) and normal rectum colon tissues ( n = 318) were analyzed based on that in the TCGA datasets through GEPIA website. (D) EBF1 mRNA expression was determined in 11 pairs of CRC samples using Q-PCR and was normalized to GAPDH. T, colorectal cancer tissues; N, non-cancerous colorectal tissues. * P

    Journal: Frontiers in Oncology

    Article Title: Transcription Factor EBF1 Over-Expression Suppresses Tumor Growth in vivo and in vitro via Modulation of the PNO1/p53 Pathway in Colorectal Cancer

    doi: 10.3389/fonc.2020.01035

    Figure Lengend Snippet: EBF1 is down-regulated in human colorectal cancer. (A) EBF1 mRNA expression (probe1: 11726800_at; probe2: 11726801_at; probe3: 11726802_a_at) in 14 matched CRC and non-cancerous colorectal tissues from our previously described gene expression profile microarrays (GEO Submission: GSE113513). Each dot represents 1 tissue. (B,C) EBF1 mRNA expression in (B) COAD tissues ( n = 279) and normal colon tissues ( n = 345), (C) READ tissues ( n = 92) and normal rectum colon tissues ( n = 318) were analyzed based on that in the TCGA datasets through GEPIA website. (D) EBF1 mRNA expression was determined in 11 pairs of CRC samples using Q-PCR and was normalized to GAPDH. T, colorectal cancer tissues; N, non-cancerous colorectal tissues. * P

    Article Snippet: Generation of Stable Transduction Cell Lines To generate a cell line stably over-expressing EBF1, HCT-116, or HT-29 cells were seeded in six-well plates (5 × 104 cells/well) and transduced with a lentiviral vector encoding full-length human EBF1 (coding region of 756 bp; Shanghai, GeneChem) or empty vector for 72 h. Transduced cells were further selected for 2 weeks using puromycin (Thermo Fisher Scientific) at 1 μg/ml.

    Techniques: Expressing, Polymerase Chain Reaction