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  • 99
    Thermo Fisher fragmented cdna
    Fragmented Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript il 7rα complementary dna cdna fragments
    Il 7rα Complementary Dna Cdna Fragments, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    TaKaRa complementary dna cdna fragments
    Analysis of <t>RAB22</t> expression. ( Upper ) Total RNA from multiple mouse tissues was probed with the full-length RAB22 <t>cDNA.</t> ( Lower ) Relative amount of total RNA in each lane is demonstrated by etidium bromide staining of 28S and 18S rRNA.
    Complementary Dna Cdna Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 89/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Addgene inc complementary dna cdna fragment
    Analysis of <t>RAB22</t> expression. ( Upper ) Total RNA from multiple mouse tissues was probed with the full-length RAB22 <t>cDNA.</t> ( Lower ) Relative amount of total RNA in each lane is demonstrated by etidium bromide staining of 28S and 18S rRNA.
    Complementary Dna Cdna Fragment, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Illumina Inc complementary dna cdna fragments
    Divergent primers detect circular RNAs in <t>cDNA</t> but not genomic <t>DNA</t> (gDNA).
    Complementary Dna Cdna Fragments, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 89/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GenScript complementary dna fragments
    <t>CREB</t> inhibits MYCT1 transcription. Notes: ( A ) Transcription factor CREB binding sites present in the MYCT1 promoter region. ( B ) MYCT1 promoter activity analysis. Hep2 cells were transfected with pGL3-P760 or pGL3-P795, followed by a luciferase reporter assay. Firefly luciferase activity was normalized to Renilla luciferase activity. ( C ) Effect of CREB on pGL3-P795 activity. pcDNA3.1 vector or pcDNA3.1-CREB was co-transfected with pGL3-P795 into Hep2 cells, followed by a luciferase reporter assay. ( D ) Effect of CREB on MYCT1 mRNA level. MYCT1 mRNA level was detected by qRT-PCR in Hep2 cells transfected with pcDNA3.1 or pcDNA3.1-CREB. ( E ) Effect of CREB on MYCT1 protein level. MYCT1 protein level was examined by Western blot in Hep2 cells transfected with pcDNA3.1 or pcDNA3.1-CREB. ( F ) Binding of CREB to MYCT1 in vivo. <t>DNA</t> fragments from Hep2 cells were amplified by PCR based on immunoprecipitation with anti-IgG or anti-CREB antibodies. ** P
    Complementary Dna Fragments, supplied by GenScript, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare cdna fragments
    The AIS of PCs is mostly covered by astrocytic processes. A , Triple immunofluorescence for GLT-1 (green; marker for astrocytes), Car8 (blue; PCs), and PV (red; PCs and BCs) is displayed in three combinations of fluorescent image pairs ( A1 , GLT1 and Car8; A2 , PV and Car8; A3 , GLT1 and PV). Arrows indicate PC axons descending in the pinceau formation. B , Immunoblot showing the specificity of GABA transporter <t>GAT-1</t> antibodies raised against two sequences (1–46 and 564–599 aa residues of mouse GAT-1). Both antibodies recognize single protein bands at 75 kDa in mouse brain homogenates (left lane) and HEK cell lysates transfected with mouse GAT-1 <t>cDNA</t> (middle) but not mouse GAT-3 (right) cDNA. Presumably because of different protein modifications, GAT-1 expressed in HEK cells are detected as two major bands at 60 and 100 kDa. C , Immunofluorescence with rabbit GAT-1 (1–46) antibody. Intense labeling is found in putative BC axons surrounding PC somata and in the pinceau formation. Asterisks indicate negative silhouettes of PC somata. Similar patterns of immunofluorescence were reproduced using rabbit GAT-1 (564–599) antibody (data not shown). D , Double immunofluorescence for GAT-1 (green) and GLT-1 (red) in the pinceau formation. Arrow indicates a PC axon descending in the pinceau formation. E , Consecutive images from preembedded immunoelectron microscopy for GLT-1 in the pinceau formation. The surface of the AIS is primarily covered by thin processes with low or negative GLT-1 labeling (filled arrowheads), which are continuous in adjacent sections to electron-lucent astrocytic processes with heavy GLT-1 labeling (open arrowheads). Scale bars: A , C , D , 5 μm; E , 250 nm.
    Cdna Fragments, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 821 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    GATC Biotech fragment complementary dna library
    The AIS of PCs is mostly covered by astrocytic processes. A , Triple immunofluorescence for GLT-1 (green; marker for astrocytes), Car8 (blue; PCs), and PV (red; PCs and BCs) is displayed in three combinations of fluorescent image pairs ( A1 , GLT1 and Car8; A2 , PV and Car8; A3 , GLT1 and PV). Arrows indicate PC axons descending in the pinceau formation. B , Immunoblot showing the specificity of GABA transporter <t>GAT-1</t> antibodies raised against two sequences (1–46 and 564–599 aa residues of mouse GAT-1). Both antibodies recognize single protein bands at 75 kDa in mouse brain homogenates (left lane) and HEK cell lysates transfected with mouse GAT-1 <t>cDNA</t> (middle) but not mouse GAT-3 (right) cDNA. Presumably because of different protein modifications, GAT-1 expressed in HEK cells are detected as two major bands at 60 and 100 kDa. C , Immunofluorescence with rabbit GAT-1 (1–46) antibody. Intense labeling is found in putative BC axons surrounding PC somata and in the pinceau formation. Asterisks indicate negative silhouettes of PC somata. Similar patterns of immunofluorescence were reproduced using rabbit GAT-1 (564–599) antibody (data not shown). D , Double immunofluorescence for GAT-1 (green) and GLT-1 (red) in the pinceau formation. Arrow indicates a PC axon descending in the pinceau formation. E , Consecutive images from preembedded immunoelectron microscopy for GLT-1 in the pinceau formation. The surface of the AIS is primarily covered by thin processes with low or negative GLT-1 labeling (filled arrowheads), which are continuous in adjacent sections to electron-lucent astrocytic processes with heavy GLT-1 labeling (open arrowheads). Scale bars: A , C , D , 5 μm; E , 250 nm.
    Fragment Complementary Dna Library, supplied by GATC Biotech, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cdna fragments
    The AIS of PCs is mostly covered by astrocytic processes. A , Triple immunofluorescence for GLT-1 (green; marker for astrocytes), Car8 (blue; PCs), and PV (red; PCs and BCs) is displayed in three combinations of fluorescent image pairs ( A1 , GLT1 and Car8; A2 , PV and Car8; A3 , GLT1 and PV). Arrows indicate PC axons descending in the pinceau formation. B , Immunoblot showing the specificity of GABA transporter <t>GAT-1</t> antibodies raised against two sequences (1–46 and 564–599 aa residues of mouse GAT-1). Both antibodies recognize single protein bands at 75 kDa in mouse brain homogenates (left lane) and HEK cell lysates transfected with mouse GAT-1 <t>cDNA</t> (middle) but not mouse GAT-3 (right) cDNA. Presumably because of different protein modifications, GAT-1 expressed in HEK cells are detected as two major bands at 60 and 100 kDa. C , Immunofluorescence with rabbit GAT-1 (1–46) antibody. Intense labeling is found in putative BC axons surrounding PC somata and in the pinceau formation. Asterisks indicate negative silhouettes of PC somata. Similar patterns of immunofluorescence were reproduced using rabbit GAT-1 (564–599) antibody (data not shown). D , Double immunofluorescence for GAT-1 (green) and GLT-1 (red) in the pinceau formation. Arrow indicates a PC axon descending in the pinceau formation. E , Consecutive images from preembedded immunoelectron microscopy for GLT-1 in the pinceau formation. The surface of the AIS is primarily covered by thin processes with low or negative GLT-1 labeling (filled arrowheads), which are continuous in adjacent sections to electron-lucent astrocytic processes with heavy GLT-1 labeling (open arrowheads). Scale bars: A , C , D , 5 μm; E , 250 nm.
    Cdna Fragments, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Source BioScience plc cdna fragments
    The AIS of PCs is mostly covered by astrocytic processes. A , Triple immunofluorescence for GLT-1 (green; marker for astrocytes), Car8 (blue; PCs), and PV (red; PCs and BCs) is displayed in three combinations of fluorescent image pairs ( A1 , GLT1 and Car8; A2 , PV and Car8; A3 , GLT1 and PV). Arrows indicate PC axons descending in the pinceau formation. B , Immunoblot showing the specificity of GABA transporter <t>GAT-1</t> antibodies raised against two sequences (1–46 and 564–599 aa residues of mouse GAT-1). Both antibodies recognize single protein bands at 75 kDa in mouse brain homogenates (left lane) and HEK cell lysates transfected with mouse GAT-1 <t>cDNA</t> (middle) but not mouse GAT-3 (right) cDNA. Presumably because of different protein modifications, GAT-1 expressed in HEK cells are detected as two major bands at 60 and 100 kDa. C , Immunofluorescence with rabbit GAT-1 (1–46) antibody. Intense labeling is found in putative BC axons surrounding PC somata and in the pinceau formation. Asterisks indicate negative silhouettes of PC somata. Similar patterns of immunofluorescence were reproduced using rabbit GAT-1 (564–599) antibody (data not shown). D , Double immunofluorescence for GAT-1 (green) and GLT-1 (red) in the pinceau formation. Arrow indicates a PC axon descending in the pinceau formation. E , Consecutive images from preembedded immunoelectron microscopy for GLT-1 in the pinceau formation. The surface of the AIS is primarily covered by thin processes with low or negative GLT-1 labeling (filled arrowheads), which are continuous in adjacent sections to electron-lucent astrocytic processes with heavy GLT-1 labeling (open arrowheads). Scale bars: A , C , D , 5 μm; E , 250 nm.
    Cdna Fragments, supplied by Source BioScience plc, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa cdna fragment
    (A) Northern blot. Poly(A) + RNA from MDBK cells was separated under denaturing conditions on a 0.8% agarose gel and blotted onto a Duranlon-UV membrane (Stratagene). The blot was hybridized with a <t>jiv-specific</t> probe labeled with [α- 32 P]dCTP. The numbers at the left side indicate the size of the RNA marker in kilobases. (B) <t>cDNA</t> cloning of the jiv-mRNA. In the upper part of the figure, the bar symbolizes the Jiv protein; different domains are pointed out on top as follows: TM, putative transmembrane region; J, J domain; and Jiv90, Jiv90 domain. The lines at both ends symbolize the noncoding regions of the mRNA. The relative positions of the restriction sites used to establish a cDNA clone encompassing the entire jiv-ORF are indicated. The cDNA library-derived clones cIK9 and cIK9-4 are shown as black bars. In the lower part of the figure, the RT-PCR fragments established to verify and complete the cDNA cloning are depicted as solid bars. The primers used for amplification are symbolized by arrowheads below. Primer M13 was complementary to the 5′ sequence of the oligonucleotide, which was ligated to the first-strand cDNA in order to analyze the 5′ end of the mRNA. (C) Nucleotide and deduced amino acid sequences of the cDNA representing the jiv-mRNA. Domains: putative transmembrane domain (amino acids 327 to 347); J domain (boldface; amino acids 444 to 505); Jiv90 domain (underlined; amino acids 533 to 622) with CXXCXXXH motifs (underlined and boldface). The shorter mRNA ends with nt 2402.
    Cdna Fragment, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Source BioScience plc calsyntenin cdna fragments
    (A) Northern blot. Poly(A) + RNA from MDBK cells was separated under denaturing conditions on a 0.8% agarose gel and blotted onto a Duranlon-UV membrane (Stratagene). The blot was hybridized with a <t>jiv-specific</t> probe labeled with [α- 32 P]dCTP. The numbers at the left side indicate the size of the RNA marker in kilobases. (B) <t>cDNA</t> cloning of the jiv-mRNA. In the upper part of the figure, the bar symbolizes the Jiv protein; different domains are pointed out on top as follows: TM, putative transmembrane region; J, J domain; and Jiv90, Jiv90 domain. The lines at both ends symbolize the noncoding regions of the mRNA. The relative positions of the restriction sites used to establish a cDNA clone encompassing the entire jiv-ORF are indicated. The cDNA library-derived clones cIK9 and cIK9-4 are shown as black bars. In the lower part of the figure, the RT-PCR fragments established to verify and complete the cDNA cloning are depicted as solid bars. The primers used for amplification are symbolized by arrowheads below. Primer M13 was complementary to the 5′ sequence of the oligonucleotide, which was ligated to the first-strand cDNA in order to analyze the 5′ end of the mRNA. (C) Nucleotide and deduced amino acid sequences of the cDNA representing the jiv-mRNA. Domains: putative transmembrane domain (amino acids 327 to 347); J domain (boldface; amino acids 444 to 505); Jiv90 domain (underlined; amino acids 533 to 622) with CXXCXXXH motifs (underlined and boldface). The shorter mRNA ends with nt 2402.
    Calsyntenin Cdna Fragments, supplied by Source BioScience plc, 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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    94
    Agilent technologies cdna fragment profiles
    Pipeline for single-neuron Patch-seq experiments. Step 1: A single neuron is characterized at the functional and morphological level by whole-cell patch-clamp electrophysiology and imaging. Step 2: Negative pressure is applied to the patch pipette used for electrophysiological recording, after which it is retracted from the chamber batch with the neuron still attached. The entire neuron, including its processes, is immediately transferred into lysis buffer by breaking the glass patch pipette tip along the inside wall of an RNase-free collection tube. Successful cell capture is always confirmed by microscopic evaluation (comparison of before and after pictures). Step 3: Single-neuron mRNA, along with external reference transcripts (ERCC RNA spike-ins) present in the lysis buffer, is reverse transcribed and the <t>cDNA</t> is PCR-amplified using SMARTer chemistry. Step 4: The synthesized cDNA is subjected to a series of QC steps based on three assays: (i) expression profiling of common housekeeping genes and RNA spike-ins by quantitative real-time PCR; (ii) fluorometric quantitation of cDNA yield (Qubit); and (iii) qualitative analysis of cDNA fragment profiles and contamination check (Bioanalyzer). Inability to detect expression of housekeeping genes while detecting expression of ERCCs is a sign of failed neuron capture, whereas inability to detect ERCCs signifies failed SMARTer cDNA synthesis. Contaminated samples are characterized by abnormally high cDNA yields that appear on the fragment analyzer as a broader peak with jaggedness or multiple peaks in the electropherogram (see also Figure 3 ). Steps 5 and 6: Libraries for RNA-seq are prepared from samples passing all QC analyses and are sequenced on an Illumina HiSeq 2500 platform. Step 7: Bioinformatics processing and analysis of the resulting RNA-seq data enable identification of unique genetic signatures associated with specific neuronal (sub)types.
    Cdna Fragment Profiles, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher gapdh cdna fragment
    Pipeline for single-neuron Patch-seq experiments. Step 1: A single neuron is characterized at the functional and morphological level by whole-cell patch-clamp electrophysiology and imaging. Step 2: Negative pressure is applied to the patch pipette used for electrophysiological recording, after which it is retracted from the chamber batch with the neuron still attached. The entire neuron, including its processes, is immediately transferred into lysis buffer by breaking the glass patch pipette tip along the inside wall of an RNase-free collection tube. Successful cell capture is always confirmed by microscopic evaluation (comparison of before and after pictures). Step 3: Single-neuron mRNA, along with external reference transcripts (ERCC RNA spike-ins) present in the lysis buffer, is reverse transcribed and the <t>cDNA</t> is PCR-amplified using SMARTer chemistry. Step 4: The synthesized cDNA is subjected to a series of QC steps based on three assays: (i) expression profiling of common housekeeping genes and RNA spike-ins by quantitative real-time PCR; (ii) fluorometric quantitation of cDNA yield (Qubit); and (iii) qualitative analysis of cDNA fragment profiles and contamination check (Bioanalyzer). Inability to detect expression of housekeeping genes while detecting expression of ERCCs is a sign of failed neuron capture, whereas inability to detect ERCCs signifies failed SMARTer cDNA synthesis. Contaminated samples are characterized by abnormally high cDNA yields that appear on the fragment analyzer as a broader peak with jaggedness or multiple peaks in the electropherogram (see also Figure 3 ). Steps 5 and 6: Libraries for RNA-seq are prepared from samples passing all QC analyses and are sequenced on an Illumina HiSeq 2500 platform. Step 7: Bioinformatics processing and analysis of the resulting RNA-seq data enable identification of unique genetic signatures associated with specific neuronal (sub)types.
    Gapdh Cdna Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GenScript synthetic cdna fragments
    Pipeline for single-neuron Patch-seq experiments. Step 1: A single neuron is characterized at the functional and morphological level by whole-cell patch-clamp electrophysiology and imaging. Step 2: Negative pressure is applied to the patch pipette used for electrophysiological recording, after which it is retracted from the chamber batch with the neuron still attached. The entire neuron, including its processes, is immediately transferred into lysis buffer by breaking the glass patch pipette tip along the inside wall of an RNase-free collection tube. Successful cell capture is always confirmed by microscopic evaluation (comparison of before and after pictures). Step 3: Single-neuron mRNA, along with external reference transcripts (ERCC RNA spike-ins) present in the lysis buffer, is reverse transcribed and the <t>cDNA</t> is PCR-amplified using SMARTer chemistry. Step 4: The synthesized cDNA is subjected to a series of QC steps based on three assays: (i) expression profiling of common housekeeping genes and RNA spike-ins by quantitative real-time PCR; (ii) fluorometric quantitation of cDNA yield (Qubit); and (iii) qualitative analysis of cDNA fragment profiles and contamination check (Bioanalyzer). Inability to detect expression of housekeeping genes while detecting expression of ERCCs is a sign of failed neuron capture, whereas inability to detect ERCCs signifies failed SMARTer cDNA synthesis. Contaminated samples are characterized by abnormally high cDNA yields that appear on the fragment analyzer as a broader peak with jaggedness or multiple peaks in the electropherogram (see also Figure 3 ). Steps 5 and 6: Libraries for RNA-seq are prepared from samples passing all QC analyses and are sequenced on an Illumina HiSeq 2500 platform. Step 7: Bioinformatics processing and analysis of the resulting RNA-seq data enable identification of unique genetic signatures associated with specific neuronal (sub)types.
    Synthetic Cdna Fragments, supplied by GenScript, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Addgene inc cdna fragment encoding gcamp3
    Pipeline for single-neuron Patch-seq experiments. Step 1: A single neuron is characterized at the functional and morphological level by whole-cell patch-clamp electrophysiology and imaging. Step 2: Negative pressure is applied to the patch pipette used for electrophysiological recording, after which it is retracted from the chamber batch with the neuron still attached. The entire neuron, including its processes, is immediately transferred into lysis buffer by breaking the glass patch pipette tip along the inside wall of an RNase-free collection tube. Successful cell capture is always confirmed by microscopic evaluation (comparison of before and after pictures). Step 3: Single-neuron mRNA, along with external reference transcripts (ERCC RNA spike-ins) present in the lysis buffer, is reverse transcribed and the <t>cDNA</t> is PCR-amplified using SMARTer chemistry. Step 4: The synthesized cDNA is subjected to a series of QC steps based on three assays: (i) expression profiling of common housekeeping genes and RNA spike-ins by quantitative real-time PCR; (ii) fluorometric quantitation of cDNA yield (Qubit); and (iii) qualitative analysis of cDNA fragment profiles and contamination check (Bioanalyzer). Inability to detect expression of housekeeping genes while detecting expression of ERCCs is a sign of failed neuron capture, whereas inability to detect ERCCs signifies failed SMARTer cDNA synthesis. Contaminated samples are characterized by abnormally high cDNA yields that appear on the fragment analyzer as a broader peak with jaggedness or multiple peaks in the electropherogram (see also Figure 3 ). Steps 5 and 6: Libraries for RNA-seq are prepared from samples passing all QC analyses and are sequenced on an Illumina HiSeq 2500 platform. Step 7: Bioinformatics processing and analysis of the resulting RNA-seq data enable identification of unique genetic signatures associated with specific neuronal (sub)types.
    Cdna Fragment Encoding Gcamp3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa human cdna fragments
    Pipeline for single-neuron Patch-seq experiments. Step 1: A single neuron is characterized at the functional and morphological level by whole-cell patch-clamp electrophysiology and imaging. Step 2: Negative pressure is applied to the patch pipette used for electrophysiological recording, after which it is retracted from the chamber batch with the neuron still attached. The entire neuron, including its processes, is immediately transferred into lysis buffer by breaking the glass patch pipette tip along the inside wall of an RNase-free collection tube. Successful cell capture is always confirmed by microscopic evaluation (comparison of before and after pictures). Step 3: Single-neuron mRNA, along with external reference transcripts (ERCC RNA spike-ins) present in the lysis buffer, is reverse transcribed and the <t>cDNA</t> is PCR-amplified using SMARTer chemistry. Step 4: The synthesized cDNA is subjected to a series of QC steps based on three assays: (i) expression profiling of common housekeeping genes and RNA spike-ins by quantitative real-time PCR; (ii) fluorometric quantitation of cDNA yield (Qubit); and (iii) qualitative analysis of cDNA fragment profiles and contamination check (Bioanalyzer). Inability to detect expression of housekeeping genes while detecting expression of ERCCs is a sign of failed neuron capture, whereas inability to detect ERCCs signifies failed SMARTer cDNA synthesis. Contaminated samples are characterized by abnormally high cDNA yields that appear on the fragment analyzer as a broader peak with jaggedness or multiple peaks in the electropherogram (see also Figure 3 ). Steps 5 and 6: Libraries for RNA-seq are prepared from samples passing all QC analyses and are sequenced on an Illumina HiSeq 2500 platform. Step 7: Bioinformatics processing and analysis of the resulting RNA-seq data enable identification of unique genetic signatures associated with specific neuronal (sub)types.
    Human Cdna Fragments, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Shanghai GenePharma atf4 cdna fragments
    Pipeline for single-neuron Patch-seq experiments. Step 1: A single neuron is characterized at the functional and morphological level by whole-cell patch-clamp electrophysiology and imaging. Step 2: Negative pressure is applied to the patch pipette used for electrophysiological recording, after which it is retracted from the chamber batch with the neuron still attached. The entire neuron, including its processes, is immediately transferred into lysis buffer by breaking the glass patch pipette tip along the inside wall of an RNase-free collection tube. Successful cell capture is always confirmed by microscopic evaluation (comparison of before and after pictures). Step 3: Single-neuron mRNA, along with external reference transcripts (ERCC RNA spike-ins) present in the lysis buffer, is reverse transcribed and the <t>cDNA</t> is PCR-amplified using SMARTer chemistry. Step 4: The synthesized cDNA is subjected to a series of QC steps based on three assays: (i) expression profiling of common housekeeping genes and RNA spike-ins by quantitative real-time PCR; (ii) fluorometric quantitation of cDNA yield (Qubit); and (iii) qualitative analysis of cDNA fragment profiles and contamination check (Bioanalyzer). Inability to detect expression of housekeeping genes while detecting expression of ERCCs is a sign of failed neuron capture, whereas inability to detect ERCCs signifies failed SMARTer cDNA synthesis. Contaminated samples are characterized by abnormally high cDNA yields that appear on the fragment analyzer as a broader peak with jaggedness or multiple peaks in the electropherogram (see also Figure 3 ). Steps 5 and 6: Libraries for RNA-seq are prepared from samples passing all QC analyses and are sequenced on an Illumina HiSeq 2500 platform. Step 7: Bioinformatics processing and analysis of the resulting RNA-seq data enable identification of unique genetic signatures associated with specific neuronal (sub)types.
    Atf4 Cdna Fragments, supplied by Shanghai GenePharma, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    atf4 cdna fragments - by Bioz Stars, 2020-04
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    86
    OriGene cav 1 cdna fragment
    Construction of recombinant chimeric virus and wild-type <t>JEV</t> <t>cDNA</t> clones. (A) For chimeric viruses, the C, prM and E genes, or just the prM and E genes of rDEN4 and rDEN4Δ30 cDNA clones (p4 and p4Δ30, respectively) were replaced with those
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    Stratagene spr 1 cdna fragment
    Construction of recombinant chimeric virus and wild-type <t>JEV</t> <t>cDNA</t> clones. (A) For chimeric viruses, the C, prM and E genes, or just the prM and E genes of rDEN4 and rDEN4Δ30 cDNA clones (p4 and p4Δ30, respectively) were replaced with those
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    GE Healthcare cdna fragment
    RU 486 loses partial agonist activity in the context of the GR dimerization-deficient mutant. (A) The GR dim mutant does not affect cell proliferation in the presence of RU 486. SAOS2-dim cells were seeded on day 0 into six-well plates (20,000 cells/well) in duplicate and cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486. Total numbers of viable cells were determined on the indicated days. (B) The RU 486-activated GR dimerization-deficient mutant fails to alter the expression of GR, CAS, or p27. SAOS2-dim cells were cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 3 days, and expression of GR, CAS, p27, and ERK in the whole-cell extracts was examined by immunoblotting. (C) The RU 486-activated dimer mutant does not induce the steady-state level of <t>p21</t> mRNA. SAOS2-dim cells were treated with an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 2 h 30 min, and total RNA was isolated and hybridized to a 32 P-labeled p21 <t>cDNA</t> probe (top panel). Equal loading into each lane is demonstrated by ethidium bromide staining of 28S rRNA (bottom panel).
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    Genewiz cdna fragment
    RU 486 loses partial agonist activity in the context of the GR dimerization-deficient mutant. (A) The GR dim mutant does not affect cell proliferation in the presence of RU 486. SAOS2-dim cells were seeded on day 0 into six-well plates (20,000 cells/well) in duplicate and cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486. Total numbers of viable cells were determined on the indicated days. (B) The RU 486-activated GR dimerization-deficient mutant fails to alter the expression of GR, CAS, or p27. SAOS2-dim cells were cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 3 days, and expression of GR, CAS, p27, and ERK in the whole-cell extracts was examined by immunoblotting. (C) The RU 486-activated dimer mutant does not induce the steady-state level of <t>p21</t> mRNA. SAOS2-dim cells were treated with an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 2 h 30 min, and total RNA was isolated and hybridized to a 32 P-labeled p21 <t>cDNA</t> probe (top panel). Equal loading into each lane is demonstrated by ethidium bromide staining of 28S rRNA (bottom panel).
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    GenScript cdna fragment
    RU 486 loses partial agonist activity in the context of the GR dimerization-deficient mutant. (A) The GR dim mutant does not affect cell proliferation in the presence of RU 486. SAOS2-dim cells were seeded on day 0 into six-well plates (20,000 cells/well) in duplicate and cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486. Total numbers of viable cells were determined on the indicated days. (B) The RU 486-activated GR dimerization-deficient mutant fails to alter the expression of GR, CAS, or p27. SAOS2-dim cells were cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 3 days, and expression of GR, CAS, p27, and ERK in the whole-cell extracts was examined by immunoblotting. (C) The RU 486-activated dimer mutant does not induce the steady-state level of <t>p21</t> mRNA. SAOS2-dim cells were treated with an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 2 h 30 min, and total RNA was isolated and hybridized to a 32 P-labeled p21 <t>cDNA</t> probe (top panel). Equal loading into each lane is demonstrated by ethidium bromide staining of 28S rRNA (bottom panel).
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    Roche cdna fragment
    Schematic representation of Tobacco rattle virus <t>(TRV)</t> and Potato virus X (PVX) constructs used in this work. ( A ) TRV is the viral vector containing the infectious TRV <t>cDNA.</t> In TRVp22 the Tomato chlorosis virus (ToCV) p22 gene was inserted into a multiple cloning site (Mcs) on the viral RNA2 created after removing the two genes involved in nematode transmission of TRV. TRV∆16K consists of an infectious TRV cDNA that harbors a premature stop codon in the RNA1-encoded 16K open reading frame (ORF) (arrowhead). In TRV∆16Kp22, the ToCV p22 gene was inserted into the Mcs and a premature stop codon was created in the RNA1 encoded 16K ORF (arrowhead); ( B ) PVX is the viral vector containing the infectious PVX cDNA. PVXp22 consists of an infectious PVX cDNA clone that expresses ToCV p22 from a duplicated PVX CP promoter. PVX∆P25 consists of an infectious PVX cDNA clone harboring two stop codons in the P25 ORF (arrowhead). PVX∆P25p22 consists of an infectious PVX cDNA clone that expresses ToCV p22 and harbors two stop codons in the P25 ORF (arrowhead). A vertical black line shows the multiple cloning site.
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    Stratagene cdna fragment
    (A) Schematic representation of the <t>VAV-3</t> cDNAs isolated in this study. Clones obtained from the screening of <t>cDNA</t> libraries are shown in regular typeface. Those obtained from RACE amplifications are shown in italics. The 5′ UTRs are shown as closed boxes. The 3′ UTRs are shown as open boxes and not in scale. The 5′ ends encoding the Vav-3 CH domain are shown as shaded boxes. Stop codons are indicated by asterisks. Start codons are indicated by inverted triangles. Important restriction sites for cloning purposes are indicated. (B) Alignment of the amino acid sequences of the three members of the Vav family in Homo sapiens . Identical residues present in the three proteins are shown on boldface. The individual structural domains are boxed. PRR, proline-rich region.
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    Agilent technologies cdna fragments
    Dissection, single-cell isolation, and genome-wide transcriptional profiling of early embryonic mouse heart. Hearts from developing embryos at indicated developmental stages were harvested and dissected into zones as shown. Single cells from each zone underwent lysis, <t>RNA</t> extraction, and <t>cDNA</t> library generation before sequencing and bioinformatics analysis. Workflow for generation and analysis of scRNA-seq data Whole-mount microscopy of murine e8.5, 9.5, and 10.5 heart showing dissection zone boundaries. IFT - inflow tract, A – atrium, V – ventricle, RA – right atrium, LA – left atrium, AVC – atrioventricular canal, RV – right ventricle, RS – right ventricular septum, LS – left ventricular septum, LV – left ventricle, OFT – outflow tract, PO – proximal outflow tract, DO – distal outflow tract. Scale bars = 500 μm. Validation of previously reported chamber-specific genes in dissected e10.5 heart zones by bulk-tissue qPCR. t-SNE plots showing major clusters among all scRNA-seq samples at each stage of development. Color and number labeling indicate zone of origin for each cell. Due to the different number of zones and cells present at each stage, t-SNE plots of different stages should not be directly compared. I – inflow tract. )
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    LGC Genomics GmbH cdna fragments
    Dissection, single-cell isolation, and genome-wide transcriptional profiling of early embryonic mouse heart. Hearts from developing embryos at indicated developmental stages were harvested and dissected into zones as shown. Single cells from each zone underwent lysis, <t>RNA</t> extraction, and <t>cDNA</t> library generation before sequencing and bioinformatics analysis. Workflow for generation and analysis of scRNA-seq data Whole-mount microscopy of murine e8.5, 9.5, and 10.5 heart showing dissection zone boundaries. IFT - inflow tract, A – atrium, V – ventricle, RA – right atrium, LA – left atrium, AVC – atrioventricular canal, RV – right ventricle, RS – right ventricular septum, LS – left ventricular septum, LV – left ventricle, OFT – outflow tract, PO – proximal outflow tract, DO – distal outflow tract. Scale bars = 500 μm. Validation of previously reported chamber-specific genes in dissected e10.5 heart zones by bulk-tissue qPCR. t-SNE plots showing major clusters among all scRNA-seq samples at each stage of development. Color and number labeling indicate zone of origin for each cell. Due to the different number of zones and cells present at each stage, t-SNE plots of different stages should not be directly compared. I – inflow tract. )
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    Medicago cdna fragments
    Dissection, single-cell isolation, and genome-wide transcriptional profiling of early embryonic mouse heart. Hearts from developing embryos at indicated developmental stages were harvested and dissected into zones as shown. Single cells from each zone underwent lysis, <t>RNA</t> extraction, and <t>cDNA</t> library generation before sequencing and bioinformatics analysis. Workflow for generation and analysis of scRNA-seq data Whole-mount microscopy of murine e8.5, 9.5, and 10.5 heart showing dissection zone boundaries. IFT - inflow tract, A – atrium, V – ventricle, RA – right atrium, LA – left atrium, AVC – atrioventricular canal, RV – right ventricle, RS – right ventricular septum, LS – left ventricular septum, LV – left ventricle, OFT – outflow tract, PO – proximal outflow tract, DO – distal outflow tract. Scale bars = 500 μm. Validation of previously reported chamber-specific genes in dissected e10.5 heart zones by bulk-tissue qPCR. t-SNE plots showing major clusters among all scRNA-seq samples at each stage of development. Color and number labeling indicate zone of origin for each cell. Due to the different number of zones and cells present at each stage, t-SNE plots of different stages should not be directly compared. I – inflow tract. )
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    Roche cdna fragments
    <t>CDP/</t> cut is regulated by PCAF HAT activity through sequences between the C3 and HD and corresponds to the acetylation of CDP/ cut in vivo . ( A ) Schematic diagram depicts various deletion mutants of the CDP/ cut <t>cDNA</t> fused in frame to the DBD of GAL4. The deletion mutants of CDP/ cut cDNA were cloned into parental plasmid pM (CLONTECH) containing the DBD of GAL4. The human CDP/ cut cDNA fragments, fused to GAL4, were tested in a transactivation assay and monitored for the repression of the cotransfected target CAT reporter gene, GAL4InrCAT, as shown in the photo ( Right ). The NULL ( A Upper ) sample corresponds to the background expression of the reporter gene construct GAL4InrCAT. ( B ) Comparison of the relative activities of the individual CDP/ cut cDNAs shown ( A ) fused to the GAL4 DBD is shown in the presence (or absence) of the cotransfected PCAF HAT activity. Results compare the ability of specific CDP/ cut deletions to repress activity of the GAL4InrCAT target. Full-length wild-type CDP/ cut [pG4-CDP (wild type)] and deletion mutant of CDP/ cut (pG4-CDPΔ1186–1256) are compared with the background level of CAT activity rendered in the presence of the parental GAL4 expression vector shown as pGAL4 ( n = 10). ( C ) To analyze the in vivo acetylation of GAL4 derived fusion proteins of CDP/ cut , G4-CDP (wild type) and G4/CDPΔ1186–1256, 8 μg of PCAF (wild type) plasmid was cotransfected with 5 μg of either pG4-CDP (wild type) or pG4/CDPΔ1186–1256 into 293 cells. Acetylation of the CDP/ cut fusions with the DBD of the GAL4 protein was determined by immunoprecipitation of equivalent amounts of G4/CDP (wild type) and G4/CDPΔ1186–1256 protein with a mouse monoclonal anti-GAL4 antibody and precipitated with goat anti-mouse IgG agarose. The immunoprecipitates, separated by SDS/PAGE, were immunoblotted with antiacetylated lysine antibodies ( Upper ). A corresponding immunoblot ( Lower ) by using an anti-GAL4 mAb was performed with the identical cell lysates [as shown ( Upper )] from 293 cells, cotransfected with PCAF and either the parental GAL4 vector pM, pG4-CDPΔ1186–1256, or pG4-CDP (wild type).
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    <t>CDP/</t> cut is regulated by PCAF HAT activity through sequences between the C3 and HD and corresponds to the acetylation of CDP/ cut in vivo . ( A ) Schematic diagram depicts various deletion mutants of the CDP/ cut <t>cDNA</t> fused in frame to the DBD of GAL4. The deletion mutants of CDP/ cut cDNA were cloned into parental plasmid pM (CLONTECH) containing the DBD of GAL4. The human CDP/ cut cDNA fragments, fused to GAL4, were tested in a transactivation assay and monitored for the repression of the cotransfected target CAT reporter gene, GAL4InrCAT, as shown in the photo ( Right ). The NULL ( A Upper ) sample corresponds to the background expression of the reporter gene construct GAL4InrCAT. ( B ) Comparison of the relative activities of the individual CDP/ cut cDNAs shown ( A ) fused to the GAL4 DBD is shown in the presence (or absence) of the cotransfected PCAF HAT activity. Results compare the ability of specific CDP/ cut deletions to repress activity of the GAL4InrCAT target. Full-length wild-type CDP/ cut [pG4-CDP (wild type)] and deletion mutant of CDP/ cut (pG4-CDPΔ1186–1256) are compared with the background level of CAT activity rendered in the presence of the parental GAL4 expression vector shown as pGAL4 ( n = 10). ( C ) To analyze the in vivo acetylation of GAL4 derived fusion proteins of CDP/ cut , G4-CDP (wild type) and G4/CDPΔ1186–1256, 8 μg of PCAF (wild type) plasmid was cotransfected with 5 μg of either pG4-CDP (wild type) or pG4/CDPΔ1186–1256 into 293 cells. Acetylation of the CDP/ cut fusions with the DBD of the GAL4 protein was determined by immunoprecipitation of equivalent amounts of G4/CDP (wild type) and G4/CDPΔ1186–1256 protein with a mouse monoclonal anti-GAL4 antibody and precipitated with goat anti-mouse IgG agarose. The immunoprecipitates, separated by SDS/PAGE, were immunoblotted with antiacetylated lysine antibodies ( Upper ). A corresponding immunoblot ( Lower ) by using an anti-GAL4 mAb was performed with the identical cell lysates [as shown ( Upper )] from 293 cells, cotransfected with PCAF and either the parental GAL4 vector pM, pG4-CDPΔ1186–1256, or pG4-CDP (wild type).
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    Boehringer Mannheim cdna fragments
    <t>CDP/</t> cut is regulated by PCAF HAT activity through sequences between the C3 and HD and corresponds to the acetylation of CDP/ cut in vivo . ( A ) Schematic diagram depicts various deletion mutants of the CDP/ cut <t>cDNA</t> fused in frame to the DBD of GAL4. The deletion mutants of CDP/ cut cDNA were cloned into parental plasmid pM (CLONTECH) containing the DBD of GAL4. The human CDP/ cut cDNA fragments, fused to GAL4, were tested in a transactivation assay and monitored for the repression of the cotransfected target CAT reporter gene, GAL4InrCAT, as shown in the photo ( Right ). The NULL ( A Upper ) sample corresponds to the background expression of the reporter gene construct GAL4InrCAT. ( B ) Comparison of the relative activities of the individual CDP/ cut cDNAs shown ( A ) fused to the GAL4 DBD is shown in the presence (or absence) of the cotransfected PCAF HAT activity. Results compare the ability of specific CDP/ cut deletions to repress activity of the GAL4InrCAT target. Full-length wild-type CDP/ cut [pG4-CDP (wild type)] and deletion mutant of CDP/ cut (pG4-CDPΔ1186–1256) are compared with the background level of CAT activity rendered in the presence of the parental GAL4 expression vector shown as pGAL4 ( n = 10). ( C ) To analyze the in vivo acetylation of GAL4 derived fusion proteins of CDP/ cut , G4-CDP (wild type) and G4/CDPΔ1186–1256, 8 μg of PCAF (wild type) plasmid was cotransfected with 5 μg of either pG4-CDP (wild type) or pG4/CDPΔ1186–1256 into 293 cells. Acetylation of the CDP/ cut fusions with the DBD of the GAL4 protein was determined by immunoprecipitation of equivalent amounts of G4/CDP (wild type) and G4/CDPΔ1186–1256 protein with a mouse monoclonal anti-GAL4 antibody and precipitated with goat anti-mouse IgG agarose. The immunoprecipitates, separated by SDS/PAGE, were immunoblotted with antiacetylated lysine antibodies ( Upper ). A corresponding immunoblot ( Lower ) by using an anti-GAL4 mAb was performed with the identical cell lysates [as shown ( Upper )] from 293 cells, cotransfected with PCAF and either the parental GAL4 vector pM, pG4-CDPΔ1186–1256, or pG4-CDP (wild type).
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    Image Search Results


    Analysis of RAB22 expression. ( Upper ) Total RNA from multiple mouse tissues was probed with the full-length RAB22 cDNA. ( Lower ) Relative amount of total RNA in each lane is demonstrated by etidium bromide staining of 28S and 18S rRNA.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: RAB22 and RAB163/mouse BRCA2: Proteins that specifically interact with the RAD51 protein

    doi:

    Figure Lengend Snippet: Analysis of RAB22 expression. ( Upper ) Total RNA from multiple mouse tissues was probed with the full-length RAB22 cDNA. ( Lower ) Relative amount of total RNA in each lane is demonstrated by etidium bromide staining of 28S and 18S rRNA.

    Article Snippet: For cloning RAB22-5 we used cDNA fragments of JG4-5–RAB22, that was originally identified through the yeast two-hybrid assay, as probes to screen the phage cDNA library, For cloning RAB22-5′-6 we used a gene specific primer (RAB22-1R, 5′-tcccctgcaggtttcttctgc-3′) and a cDNA adapter primer (AP1; Clontech) to amplify the 5′ end of the RAB22 cDNA by PCR.

    Techniques: Expressing, Staining

    Divergent primers detect circular RNAs in cDNA but not genomic DNA (gDNA).

    Journal: Frontiers in Oncology

    Article Title: Circular RNA Expression in Oral Squamous Cell Carcinoma

    doi: 10.3389/fonc.2018.00398

    Figure Lengend Snippet: Divergent primers detect circular RNAs in cDNA but not genomic DNA (gDNA).

    Article Snippet: Next, the cDNA fragments were purified with VAHTSTM DNA Clean Beads, end repaired, poly (A) added, and ligated to Illumina sequencing adapters.

    Techniques:

    CREB inhibits MYCT1 transcription. Notes: ( A ) Transcription factor CREB binding sites present in the MYCT1 promoter region. ( B ) MYCT1 promoter activity analysis. Hep2 cells were transfected with pGL3-P760 or pGL3-P795, followed by a luciferase reporter assay. Firefly luciferase activity was normalized to Renilla luciferase activity. ( C ) Effect of CREB on pGL3-P795 activity. pcDNA3.1 vector or pcDNA3.1-CREB was co-transfected with pGL3-P795 into Hep2 cells, followed by a luciferase reporter assay. ( D ) Effect of CREB on MYCT1 mRNA level. MYCT1 mRNA level was detected by qRT-PCR in Hep2 cells transfected with pcDNA3.1 or pcDNA3.1-CREB. ( E ) Effect of CREB on MYCT1 protein level. MYCT1 protein level was examined by Western blot in Hep2 cells transfected with pcDNA3.1 or pcDNA3.1-CREB. ( F ) Binding of CREB to MYCT1 in vivo. DNA fragments from Hep2 cells were amplified by PCR based on immunoprecipitation with anti-IgG or anti-CREB antibodies. ** P

    Journal: OncoTargets and therapy

    Article Title: CREB promotes laryngeal cancer cell migration via MYCT1/NAT10 axis

    doi: 10.2147/OTT.S156582

    Figure Lengend Snippet: CREB inhibits MYCT1 transcription. Notes: ( A ) Transcription factor CREB binding sites present in the MYCT1 promoter region. ( B ) MYCT1 promoter activity analysis. Hep2 cells were transfected with pGL3-P760 or pGL3-P795, followed by a luciferase reporter assay. Firefly luciferase activity was normalized to Renilla luciferase activity. ( C ) Effect of CREB on pGL3-P795 activity. pcDNA3.1 vector or pcDNA3.1-CREB was co-transfected with pGL3-P795 into Hep2 cells, followed by a luciferase reporter assay. ( D ) Effect of CREB on MYCT1 mRNA level. MYCT1 mRNA level was detected by qRT-PCR in Hep2 cells transfected with pcDNA3.1 or pcDNA3.1-CREB. ( E ) Effect of CREB on MYCT1 protein level. MYCT1 protein level was examined by Western blot in Hep2 cells transfected with pcDNA3.1 or pcDNA3.1-CREB. ( F ) Binding of CREB to MYCT1 in vivo. DNA fragments from Hep2 cells were amplified by PCR based on immunoprecipitation with anti-IgG or anti-CREB antibodies. ** P

    Article Snippet: Complementary DNA fragments encoding human CREB, MYCT1, and NAT10 were constructed into pcDNA3.1 vector (GenScript Company).

    Techniques: Binding Assay, Activity Assay, Transfection, Luciferase, Reporter Assay, Plasmid Preparation, Quantitative RT-PCR, Western Blot, In Vivo, Amplification, Polymerase Chain Reaction, Immunoprecipitation

    The AIS of PCs is mostly covered by astrocytic processes. A , Triple immunofluorescence for GLT-1 (green; marker for astrocytes), Car8 (blue; PCs), and PV (red; PCs and BCs) is displayed in three combinations of fluorescent image pairs ( A1 , GLT1 and Car8; A2 , PV and Car8; A3 , GLT1 and PV). Arrows indicate PC axons descending in the pinceau formation. B , Immunoblot showing the specificity of GABA transporter GAT-1 antibodies raised against two sequences (1–46 and 564–599 aa residues of mouse GAT-1). Both antibodies recognize single protein bands at 75 kDa in mouse brain homogenates (left lane) and HEK cell lysates transfected with mouse GAT-1 cDNA (middle) but not mouse GAT-3 (right) cDNA. Presumably because of different protein modifications, GAT-1 expressed in HEK cells are detected as two major bands at 60 and 100 kDa. C , Immunofluorescence with rabbit GAT-1 (1–46) antibody. Intense labeling is found in putative BC axons surrounding PC somata and in the pinceau formation. Asterisks indicate negative silhouettes of PC somata. Similar patterns of immunofluorescence were reproduced using rabbit GAT-1 (564–599) antibody (data not shown). D , Double immunofluorescence for GAT-1 (green) and GLT-1 (red) in the pinceau formation. Arrow indicates a PC axon descending in the pinceau formation. E , Consecutive images from preembedded immunoelectron microscopy for GLT-1 in the pinceau formation. The surface of the AIS is primarily covered by thin processes with low or negative GLT-1 labeling (filled arrowheads), which are continuous in adjacent sections to electron-lucent astrocytic processes with heavy GLT-1 labeling (open arrowheads). Scale bars: A , C , D , 5 μm; E , 250 nm.

    Journal: The Journal of Neuroscience

    Article Title: Lack of Molecular–Anatomical Evidence for GABAergic Influence on Axon Initial Segment of Cerebellar Purkinje Cells by the Pinceau Formation

    doi: 10.1523/JNEUROSCI.1651-12.2012

    Figure Lengend Snippet: The AIS of PCs is mostly covered by astrocytic processes. A , Triple immunofluorescence for GLT-1 (green; marker for astrocytes), Car8 (blue; PCs), and PV (red; PCs and BCs) is displayed in three combinations of fluorescent image pairs ( A1 , GLT1 and Car8; A2 , PV and Car8; A3 , GLT1 and PV). Arrows indicate PC axons descending in the pinceau formation. B , Immunoblot showing the specificity of GABA transporter GAT-1 antibodies raised against two sequences (1–46 and 564–599 aa residues of mouse GAT-1). Both antibodies recognize single protein bands at 75 kDa in mouse brain homogenates (left lane) and HEK cell lysates transfected with mouse GAT-1 cDNA (middle) but not mouse GAT-3 (right) cDNA. Presumably because of different protein modifications, GAT-1 expressed in HEK cells are detected as two major bands at 60 and 100 kDa. C , Immunofluorescence with rabbit GAT-1 (1–46) antibody. Intense labeling is found in putative BC axons surrounding PC somata and in the pinceau formation. Asterisks indicate negative silhouettes of PC somata. Similar patterns of immunofluorescence were reproduced using rabbit GAT-1 (564–599) antibody (data not shown). D , Double immunofluorescence for GAT-1 (green) and GLT-1 (red) in the pinceau formation. Arrow indicates a PC axon descending in the pinceau formation. E , Consecutive images from preembedded immunoelectron microscopy for GLT-1 in the pinceau formation. The surface of the AIS is primarily covered by thin processes with low or negative GLT-1 labeling (filled arrowheads), which are continuous in adjacent sections to electron-lucent astrocytic processes with heavy GLT-1 labeling (open arrowheads). Scale bars: A , C , D , 5 μm; E , 250 nm.

    Article Snippet: Glutathione S -transferase fusion proteins were used for antigens of GAT-1 and NL2 antibodies by subcloning their cDNA fragments into the BamHI/EcoRI site of the pGEX4T-2 plasmid (GE Healthcare).

    Techniques: Immunofluorescence, Marker, Transfection, Labeling, Immuno-Electron Microscopy

    Immunofluorescence showing the lack of postsynaptic molecules required for GABAergic synapses on the AIS of PCs. A , Triple immunofluorescence for AnkG (green; A1 ), Car8 (blue; A1 ), and GABA A Rα1 (red; A1 , A2 ) in PCs. Note that GABA A Rα1-immunoreactive clusters are found on Car8-labeled somata or the axon hillock of PCs (arrowheads) but not on the AIS colabeled for AnkG and Car8 (arrows). A doubled arrowhead indicates a GABA A Rα1 cluster on an AnkG-lacking portion of PC axon. B , Immunoblot showing the specificity of rabbit NL2 antibody. Single protein bands at 100 kDa are detected in HEK cell lysates transfected (left lane) with NL2 cDNA and mouse brain homogenates (right lane) but not in nontransfected HEK cell lysates (mock, middle lane). C , Double immunofluorescence for NL2 (red) and GAD (green). Extensive colocalization in the neuropil and around PC somata supports the specificity of NL2 immunolabeling. D , Triple immunofluorescence for AnkG (green), Car8 (blue), and NL2 (red) in PCs. Note that NL2 + puncta are detected on Car8-labeled PC somata (arrowheads) but not on AnkG-labeled AISs (arrow). Scale bars: 5 μm; C , D , 10 μm.

    Journal: The Journal of Neuroscience

    Article Title: Lack of Molecular–Anatomical Evidence for GABAergic Influence on Axon Initial Segment of Cerebellar Purkinje Cells by the Pinceau Formation

    doi: 10.1523/JNEUROSCI.1651-12.2012

    Figure Lengend Snippet: Immunofluorescence showing the lack of postsynaptic molecules required for GABAergic synapses on the AIS of PCs. A , Triple immunofluorescence for AnkG (green; A1 ), Car8 (blue; A1 ), and GABA A Rα1 (red; A1 , A2 ) in PCs. Note that GABA A Rα1-immunoreactive clusters are found on Car8-labeled somata or the axon hillock of PCs (arrowheads) but not on the AIS colabeled for AnkG and Car8 (arrows). A doubled arrowhead indicates a GABA A Rα1 cluster on an AnkG-lacking portion of PC axon. B , Immunoblot showing the specificity of rabbit NL2 antibody. Single protein bands at 100 kDa are detected in HEK cell lysates transfected (left lane) with NL2 cDNA and mouse brain homogenates (right lane) but not in nontransfected HEK cell lysates (mock, middle lane). C , Double immunofluorescence for NL2 (red) and GAD (green). Extensive colocalization in the neuropil and around PC somata supports the specificity of NL2 immunolabeling. D , Triple immunofluorescence for AnkG (green), Car8 (blue), and NL2 (red) in PCs. Note that NL2 + puncta are detected on Car8-labeled PC somata (arrowheads) but not on AnkG-labeled AISs (arrow). Scale bars: 5 μm; C , D , 10 μm.

    Article Snippet: Glutathione S -transferase fusion proteins were used for antigens of GAT-1 and NL2 antibodies by subcloning their cDNA fragments into the BamHI/EcoRI site of the pGEX4T-2 plasmid (GE Healthcare).

    Techniques: Immunofluorescence, Labeling, Transfection, Immunolabeling

    (A) Northern blot. Poly(A) + RNA from MDBK cells was separated under denaturing conditions on a 0.8% agarose gel and blotted onto a Duranlon-UV membrane (Stratagene). The blot was hybridized with a jiv-specific probe labeled with [α- 32 P]dCTP. The numbers at the left side indicate the size of the RNA marker in kilobases. (B) cDNA cloning of the jiv-mRNA. In the upper part of the figure, the bar symbolizes the Jiv protein; different domains are pointed out on top as follows: TM, putative transmembrane region; J, J domain; and Jiv90, Jiv90 domain. The lines at both ends symbolize the noncoding regions of the mRNA. The relative positions of the restriction sites used to establish a cDNA clone encompassing the entire jiv-ORF are indicated. The cDNA library-derived clones cIK9 and cIK9-4 are shown as black bars. In the lower part of the figure, the RT-PCR fragments established to verify and complete the cDNA cloning are depicted as solid bars. The primers used for amplification are symbolized by arrowheads below. Primer M13 was complementary to the 5′ sequence of the oligonucleotide, which was ligated to the first-strand cDNA in order to analyze the 5′ end of the mRNA. (C) Nucleotide and deduced amino acid sequences of the cDNA representing the jiv-mRNA. Domains: putative transmembrane domain (amino acids 327 to 347); J domain (boldface; amino acids 444 to 505); Jiv90 domain (underlined; amino acids 533 to 622) with CXXCXXXH motifs (underlined and boldface). The shorter mRNA ends with nt 2402.

    Journal: Journal of Virology

    Article Title: A Cellular J-Domain Protein Modulates Polyprotein Processing and Cytopathogenicity of a Pestivirus

    doi: 10.1128/JVI.75.19.9470-9482.2001

    Figure Lengend Snippet: (A) Northern blot. Poly(A) + RNA from MDBK cells was separated under denaturing conditions on a 0.8% agarose gel and blotted onto a Duranlon-UV membrane (Stratagene). The blot was hybridized with a jiv-specific probe labeled with [α- 32 P]dCTP. The numbers at the left side indicate the size of the RNA marker in kilobases. (B) cDNA cloning of the jiv-mRNA. In the upper part of the figure, the bar symbolizes the Jiv protein; different domains are pointed out on top as follows: TM, putative transmembrane region; J, J domain; and Jiv90, Jiv90 domain. The lines at both ends symbolize the noncoding regions of the mRNA. The relative positions of the restriction sites used to establish a cDNA clone encompassing the entire jiv-ORF are indicated. The cDNA library-derived clones cIK9 and cIK9-4 are shown as black bars. In the lower part of the figure, the RT-PCR fragments established to verify and complete the cDNA cloning are depicted as solid bars. The primers used for amplification are symbolized by arrowheads below. Primer M13 was complementary to the 5′ sequence of the oligonucleotide, which was ligated to the first-strand cDNA in order to analyze the 5′ end of the mRNA. (C) Nucleotide and deduced amino acid sequences of the cDNA representing the jiv-mRNA. Domains: putative transmembrane domain (amino acids 327 to 347); J domain (boldface; amino acids 444 to 505); Jiv90 domain (underlined; amino acids 533 to 622) with CXXCXXXH motifs (underlined and boldface). The shorter mRNA ends with nt 2402.

    Article Snippet: A cDNA fragment encompassing the entire Jiv-ORF was ligated into plasmid pTRE (Clontech, Palo Alto, Calif.).

    Techniques: Northern Blot, Agarose Gel Electrophoresis, Labeling, Marker, Clone Assay, cDNA Library Assay, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing

    Pipeline for single-neuron Patch-seq experiments. Step 1: A single neuron is characterized at the functional and morphological level by whole-cell patch-clamp electrophysiology and imaging. Step 2: Negative pressure is applied to the patch pipette used for electrophysiological recording, after which it is retracted from the chamber batch with the neuron still attached. The entire neuron, including its processes, is immediately transferred into lysis buffer by breaking the glass patch pipette tip along the inside wall of an RNase-free collection tube. Successful cell capture is always confirmed by microscopic evaluation (comparison of before and after pictures). Step 3: Single-neuron mRNA, along with external reference transcripts (ERCC RNA spike-ins) present in the lysis buffer, is reverse transcribed and the cDNA is PCR-amplified using SMARTer chemistry. Step 4: The synthesized cDNA is subjected to a series of QC steps based on three assays: (i) expression profiling of common housekeeping genes and RNA spike-ins by quantitative real-time PCR; (ii) fluorometric quantitation of cDNA yield (Qubit); and (iii) qualitative analysis of cDNA fragment profiles and contamination check (Bioanalyzer). Inability to detect expression of housekeeping genes while detecting expression of ERCCs is a sign of failed neuron capture, whereas inability to detect ERCCs signifies failed SMARTer cDNA synthesis. Contaminated samples are characterized by abnormally high cDNA yields that appear on the fragment analyzer as a broader peak with jaggedness or multiple peaks in the electropherogram (see also Figure 3 ). Steps 5 and 6: Libraries for RNA-seq are prepared from samples passing all QC analyses and are sequenced on an Illumina HiSeq 2500 platform. Step 7: Bioinformatics processing and analysis of the resulting RNA-seq data enable identification of unique genetic signatures associated with specific neuronal (sub)types.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Patch-Seq Protocol to Analyze the Electrophysiology, Morphology and Transcriptome of Whole Single Neurons Derived From Human Pluripotent Stem Cells

    doi: 10.3389/fnmol.2018.00261

    Figure Lengend Snippet: Pipeline for single-neuron Patch-seq experiments. Step 1: A single neuron is characterized at the functional and morphological level by whole-cell patch-clamp electrophysiology and imaging. Step 2: Negative pressure is applied to the patch pipette used for electrophysiological recording, after which it is retracted from the chamber batch with the neuron still attached. The entire neuron, including its processes, is immediately transferred into lysis buffer by breaking the glass patch pipette tip along the inside wall of an RNase-free collection tube. Successful cell capture is always confirmed by microscopic evaluation (comparison of before and after pictures). Step 3: Single-neuron mRNA, along with external reference transcripts (ERCC RNA spike-ins) present in the lysis buffer, is reverse transcribed and the cDNA is PCR-amplified using SMARTer chemistry. Step 4: The synthesized cDNA is subjected to a series of QC steps based on three assays: (i) expression profiling of common housekeeping genes and RNA spike-ins by quantitative real-time PCR; (ii) fluorometric quantitation of cDNA yield (Qubit); and (iii) qualitative analysis of cDNA fragment profiles and contamination check (Bioanalyzer). Inability to detect expression of housekeeping genes while detecting expression of ERCCs is a sign of failed neuron capture, whereas inability to detect ERCCs signifies failed SMARTer cDNA synthesis. Contaminated samples are characterized by abnormally high cDNA yields that appear on the fragment analyzer as a broader peak with jaggedness or multiple peaks in the electropherogram (see also Figure 3 ). Steps 5 and 6: Libraries for RNA-seq are prepared from samples passing all QC analyses and are sequenced on an Illumina HiSeq 2500 platform. Step 7: Bioinformatics processing and analysis of the resulting RNA-seq data enable identification of unique genetic signatures associated with specific neuronal (sub)types.

    Article Snippet: The QC pipeline described here is based on (i) expression profiling of common housekeeping genes and differentially abundant exogenous reference transcripts (ERCC spike-in controls), (ii) fluorometric quantitation of cDNA yield (Qubit), and (iii) qualitative analysis of cDNA fragment profiles (Agilent Bioanalyzer).

    Techniques: Functional Assay, Patch Clamp, Imaging, Transferring, Lysis, Polymerase Chain Reaction, Amplification, Synthesized, Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, RNA Sequencing Assay

    Construction of recombinant chimeric virus and wild-type JEV cDNA clones. (A) For chimeric viruses, the C, prM and E genes, or just the prM and E genes of rDEN4 and rDEN4Δ30 cDNA clones (p4 and p4Δ30, respectively) were replaced with those

    Journal: Vaccine

    Article Title: Japanese encephalitis virus vaccine candidates generated by chimerization with dengue virus type 4

    doi: 10.1016/j.vaccine.2014.03.062

    Figure Lengend Snippet: Construction of recombinant chimeric virus and wild-type JEV cDNA clones. (A) For chimeric viruses, the C, prM and E genes, or just the prM and E genes of rDEN4 and rDEN4Δ30 cDNA clones (p4 and p4Δ30, respectively) were replaced with those

    Article Snippet: A cDNA fragment corresponding to the JEV India/78C, prM, and E genes was synthesized and cloned into mammalian expression vector pCMV6-AC (OriGene) to generate pJEV-CprME.

    Techniques: Recombinant, Clone Assay

    RU 486 loses partial agonist activity in the context of the GR dimerization-deficient mutant. (A) The GR dim mutant does not affect cell proliferation in the presence of RU 486. SAOS2-dim cells were seeded on day 0 into six-well plates (20,000 cells/well) in duplicate and cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486. Total numbers of viable cells were determined on the indicated days. (B) The RU 486-activated GR dimerization-deficient mutant fails to alter the expression of GR, CAS, or p27. SAOS2-dim cells were cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 3 days, and expression of GR, CAS, p27, and ERK in the whole-cell extracts was examined by immunoblotting. (C) The RU 486-activated dimer mutant does not induce the steady-state level of p21 mRNA. SAOS2-dim cells were treated with an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 2 h 30 min, and total RNA was isolated and hybridized to a 32 P-labeled p21 cDNA probe (top panel). Equal loading into each lane is demonstrated by ethidium bromide staining of 28S rRNA (bottom panel).

    Journal: Molecular and Cellular Biology

    Article Title: Distinct Glucocorticoid Receptor Transcriptional Regulatory Surfaces Mediate the Cytotoxic and Cytostatic Effects of Glucocorticoids

    doi:

    Figure Lengend Snippet: RU 486 loses partial agonist activity in the context of the GR dimerization-deficient mutant. (A) The GR dim mutant does not affect cell proliferation in the presence of RU 486. SAOS2-dim cells were seeded on day 0 into six-well plates (20,000 cells/well) in duplicate and cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486. Total numbers of viable cells were determined on the indicated days. (B) The RU 486-activated GR dimerization-deficient mutant fails to alter the expression of GR, CAS, or p27. SAOS2-dim cells were cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 3 days, and expression of GR, CAS, p27, and ERK in the whole-cell extracts was examined by immunoblotting. (C) The RU 486-activated dimer mutant does not induce the steady-state level of p21 mRNA. SAOS2-dim cells were treated with an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 2 h 30 min, and total RNA was isolated and hybridized to a 32 P-labeled p21 cDNA probe (top panel). Equal loading into each lane is demonstrated by ethidium bromide staining of 28S rRNA (bottom panel).

    Article Snippet: A cDNA fragment coding for p21 was labeled with [α-32 P]dCTP by using a RediPrime random priming labeling kit (Amersham) as per the manufacturer’s instructions.

    Techniques: Activity Assay, Mutagenesis, Cell Culture, Expressing, Isolation, Labeling, Staining

    Induction of CDI expression by the wt GR in SAOS2 cells is uncoupled by RU 486. (A) Differential effects of GR ligands on the proliferation of SAOS2-GR(+) cells. SAOS2-GR(+) cells were seeded on day 0 into six-well plates (20,000 cells/well) in duplicate and cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486. Total numbers of viable cells were determined on the indicated days. Note 69% inhibition of cell proliferation in the presence of RU 486, compared to 98% observed in the presence of Dex. (B) Alterations in protein expression induced by RU 486 in SAOS2-GR(+) cells. SAOS2-GR(+) cells were cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 3 days, and expression of GR, CAS, p27, Bcl2, and ERK in whole-cell extracts was examined by immunoblotting. Note that comparatively mild repression of GR and CAS is observed in the presence of RU 486, whereas p27 induction is retained whether GR is activated with Dex or RU 486. Similar results were obtained with RU 486 concentrations of 500 nM and 1 μM (data not shown). (C) RU 486-activated wt GR or LS7 mutant fails to efficiently induce p21 mRNA expression in SAOS2 cells. SAOS2-GR(+) (wt GR) and SAOS2-LS7 (LS7) cells were treated with an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 2 h 30 min, and total RNA was isolated and subjected to Northern hybridization with a 32 P-labeled p21 cDNA probe (top panels). Equal loading in each lane is demonstrated by ethidium bromide staining of 28S rRNA (bottom panels). The results of autoradiography were quantitated by spot densitometry. The densitometric value obtained for mock-treated SAOS2-GR(+) or SAOS2-LS7 cells was arbitrarily set as 1×.

    Journal: Molecular and Cellular Biology

    Article Title: Distinct Glucocorticoid Receptor Transcriptional Regulatory Surfaces Mediate the Cytotoxic and Cytostatic Effects of Glucocorticoids

    doi:

    Figure Lengend Snippet: Induction of CDI expression by the wt GR in SAOS2 cells is uncoupled by RU 486. (A) Differential effects of GR ligands on the proliferation of SAOS2-GR(+) cells. SAOS2-GR(+) cells were seeded on day 0 into six-well plates (20,000 cells/well) in duplicate and cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486. Total numbers of viable cells were determined on the indicated days. Note 69% inhibition of cell proliferation in the presence of RU 486, compared to 98% observed in the presence of Dex. (B) Alterations in protein expression induced by RU 486 in SAOS2-GR(+) cells. SAOS2-GR(+) cells were cultured in the presence of an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 3 days, and expression of GR, CAS, p27, Bcl2, and ERK in whole-cell extracts was examined by immunoblotting. Note that comparatively mild repression of GR and CAS is observed in the presence of RU 486, whereas p27 induction is retained whether GR is activated with Dex or RU 486. Similar results were obtained with RU 486 concentrations of 500 nM and 1 μM (data not shown). (C) RU 486-activated wt GR or LS7 mutant fails to efficiently induce p21 mRNA expression in SAOS2 cells. SAOS2-GR(+) (wt GR) and SAOS2-LS7 (LS7) cells were treated with an ethanol vehicle, 100 nM Dex, or 100 nM RU 486 for 2 h 30 min, and total RNA was isolated and subjected to Northern hybridization with a 32 P-labeled p21 cDNA probe (top panels). Equal loading in each lane is demonstrated by ethidium bromide staining of 28S rRNA (bottom panels). The results of autoradiography were quantitated by spot densitometry. The densitometric value obtained for mock-treated SAOS2-GR(+) or SAOS2-LS7 cells was arbitrarily set as 1×.

    Article Snippet: A cDNA fragment coding for p21 was labeled with [α-32 P]dCTP by using a RediPrime random priming labeling kit (Amersham) as per the manufacturer’s instructions.

    Techniques: Expressing, Cell Culture, Inhibition, Mutagenesis, Isolation, Northern Blot, Hybridization, Labeling, Staining, Autoradiography

    Transcriptional activation by the GR AF-1 is dispensable for cell cycle arrest in SAOS2 cells. (A) The GR 30iiB mutant induces complete cell cycle arrest in SAOS2 cells. SAOS2-30iiB stable transformants were generated as described in Materials and Methods. SAOS2-30iiB clones were seeded on day 0 into six-well plates (20,000 cells/well) and cultured in the absence or presence of 100 nM Dex. Total numbers of viable cells were determined on the indicated days by using the trypan blue exclusion method. (B) Induction of p21 and p27 by the GR 30iiB mutant. SAOS2-GR(+) (wt GR) and SAOS2-30iiB (30iiB) cells were cultured in the presence of 100 nM Dex or ethanol vehicle for 3 days, and whole-cell extracts were prepared and subjected to immunoblotting for GR, p27, p21, and ERK. Note the induction of p21 and p27 by both the wt GR and the 30iiB mutant. (C) Time course of p21 mRNA induction. SAOS2-GR(+) (wt GR) and SAOS2-30iiB (30iiB) cells were treated with 100 nM Dex for 0, 15, 30, 60, and 120 min, and total RNA was isolated and subjected to Northern blot analysis with a 32 P-labeled p21 cDNA probe (top panels). Equal loading in each lane is demonstrated by ethidium bromide staining of the 28S rRNA (bottom panels).

    Journal: Molecular and Cellular Biology

    Article Title: Distinct Glucocorticoid Receptor Transcriptional Regulatory Surfaces Mediate the Cytotoxic and Cytostatic Effects of Glucocorticoids

    doi:

    Figure Lengend Snippet: Transcriptional activation by the GR AF-1 is dispensable for cell cycle arrest in SAOS2 cells. (A) The GR 30iiB mutant induces complete cell cycle arrest in SAOS2 cells. SAOS2-30iiB stable transformants were generated as described in Materials and Methods. SAOS2-30iiB clones were seeded on day 0 into six-well plates (20,000 cells/well) and cultured in the absence or presence of 100 nM Dex. Total numbers of viable cells were determined on the indicated days by using the trypan blue exclusion method. (B) Induction of p21 and p27 by the GR 30iiB mutant. SAOS2-GR(+) (wt GR) and SAOS2-30iiB (30iiB) cells were cultured in the presence of 100 nM Dex or ethanol vehicle for 3 days, and whole-cell extracts were prepared and subjected to immunoblotting for GR, p27, p21, and ERK. Note the induction of p21 and p27 by both the wt GR and the 30iiB mutant. (C) Time course of p21 mRNA induction. SAOS2-GR(+) (wt GR) and SAOS2-30iiB (30iiB) cells were treated with 100 nM Dex for 0, 15, 30, 60, and 120 min, and total RNA was isolated and subjected to Northern blot analysis with a 32 P-labeled p21 cDNA probe (top panels). Equal loading in each lane is demonstrated by ethidium bromide staining of the 28S rRNA (bottom panels).

    Article Snippet: A cDNA fragment coding for p21 was labeled with [α-32 P]dCTP by using a RediPrime random priming labeling kit (Amersham) as per the manufacturer’s instructions.

    Techniques: Activation Assay, Mutagenesis, Generated, Clone Assay, Cell Culture, Isolation, Northern Blot, Labeling, Staining

    The GR LS7 mutant functions like the wt GR when stably expressed in SAOS2 cells. (A) Inhibition of SAOS2 cell proliferation by the GR LS7 mutant. Multiple SAOS2-LS7 clones were generated as described in Materials and Methods. SAOS2-LS7 cells were seeded on day 0 into six-well plates (20,000 cells/well) and cultured in the absence or presence of 100 nM Dex. Total numbers of viable cells were determined on the indicated days by using the trypan blue exclusion method. Similar cell growth kinetics were observed in three independent LS7-expressing clones. (B) The GR LS7 mutant induces the expression of p21 and p27 proteins. SAOS2-GR(+) (wt GR), SAOS2-LS7 (LS7), and GR-negative SAOS2 (con) cells were cultured in the presence of 100 nM Dex or ethanol vehicle for 3 days, and whole-cell extracts were prepared and subjected to immunoblotting for GR, p27, p21, and ERK. Note the increase in p21 and p27 protein in the wt GR- and LS7-expressing cells but not in the GR-deficient control cells. (C) Induction of the steady-state p21 mRNA level by the GR LS7 mutant. SAOS2-GR(+) (wt GR) and SAOS2-LS7 (LS7) cells were treated with 100 nM Dex for 2 h 30 min where indicated, and total RNA was isolated and hybridized to a 32 P-labeled p21 cDNA probe (top panel). Equal loading in each lane is demonstrated by ethidium bromide staining of the 28S rRNA (bottom panel).

    Journal: Molecular and Cellular Biology

    Article Title: Distinct Glucocorticoid Receptor Transcriptional Regulatory Surfaces Mediate the Cytotoxic and Cytostatic Effects of Glucocorticoids

    doi:

    Figure Lengend Snippet: The GR LS7 mutant functions like the wt GR when stably expressed in SAOS2 cells. (A) Inhibition of SAOS2 cell proliferation by the GR LS7 mutant. Multiple SAOS2-LS7 clones were generated as described in Materials and Methods. SAOS2-LS7 cells were seeded on day 0 into six-well plates (20,000 cells/well) and cultured in the absence or presence of 100 nM Dex. Total numbers of viable cells were determined on the indicated days by using the trypan blue exclusion method. Similar cell growth kinetics were observed in three independent LS7-expressing clones. (B) The GR LS7 mutant induces the expression of p21 and p27 proteins. SAOS2-GR(+) (wt GR), SAOS2-LS7 (LS7), and GR-negative SAOS2 (con) cells were cultured in the presence of 100 nM Dex or ethanol vehicle for 3 days, and whole-cell extracts were prepared and subjected to immunoblotting for GR, p27, p21, and ERK. Note the increase in p21 and p27 protein in the wt GR- and LS7-expressing cells but not in the GR-deficient control cells. (C) Induction of the steady-state p21 mRNA level by the GR LS7 mutant. SAOS2-GR(+) (wt GR) and SAOS2-LS7 (LS7) cells were treated with 100 nM Dex for 2 h 30 min where indicated, and total RNA was isolated and hybridized to a 32 P-labeled p21 cDNA probe (top panel). Equal loading in each lane is demonstrated by ethidium bromide staining of the 28S rRNA (bottom panel).

    Article Snippet: A cDNA fragment coding for p21 was labeled with [α-32 P]dCTP by using a RediPrime random priming labeling kit (Amersham) as per the manufacturer’s instructions.

    Techniques: Mutagenesis, Stable Transfection, Inhibition, Clone Assay, Generated, Cell Culture, Expressing, Isolation, Labeling, Staining

    Schematic representation of Tobacco rattle virus (TRV) and Potato virus X (PVX) constructs used in this work. ( A ) TRV is the viral vector containing the infectious TRV cDNA. In TRVp22 the Tomato chlorosis virus (ToCV) p22 gene was inserted into a multiple cloning site (Mcs) on the viral RNA2 created after removing the two genes involved in nematode transmission of TRV. TRV∆16K consists of an infectious TRV cDNA that harbors a premature stop codon in the RNA1-encoded 16K open reading frame (ORF) (arrowhead). In TRV∆16Kp22, the ToCV p22 gene was inserted into the Mcs and a premature stop codon was created in the RNA1 encoded 16K ORF (arrowhead); ( B ) PVX is the viral vector containing the infectious PVX cDNA. PVXp22 consists of an infectious PVX cDNA clone that expresses ToCV p22 from a duplicated PVX CP promoter. PVX∆P25 consists of an infectious PVX cDNA clone harboring two stop codons in the P25 ORF (arrowhead). PVX∆P25p22 consists of an infectious PVX cDNA clone that expresses ToCV p22 and harbors two stop codons in the P25 ORF (arrowhead). A vertical black line shows the multiple cloning site.

    Journal: Viruses

    Article Title: The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses

    doi: 10.3390/v9120358

    Figure Lengend Snippet: Schematic representation of Tobacco rattle virus (TRV) and Potato virus X (PVX) constructs used in this work. ( A ) TRV is the viral vector containing the infectious TRV cDNA. In TRVp22 the Tomato chlorosis virus (ToCV) p22 gene was inserted into a multiple cloning site (Mcs) on the viral RNA2 created after removing the two genes involved in nematode transmission of TRV. TRV∆16K consists of an infectious TRV cDNA that harbors a premature stop codon in the RNA1-encoded 16K open reading frame (ORF) (arrowhead). In TRV∆16Kp22, the ToCV p22 gene was inserted into the Mcs and a premature stop codon was created in the RNA1 encoded 16K ORF (arrowhead); ( B ) PVX is the viral vector containing the infectious PVX cDNA. PVXp22 consists of an infectious PVX cDNA clone that expresses ToCV p22 from a duplicated PVX CP promoter. PVX∆P25 consists of an infectious PVX cDNA clone harboring two stop codons in the P25 ORF (arrowhead). PVX∆P25p22 consists of an infectious PVX cDNA clone that expresses ToCV p22 and harbors two stop codons in the P25 ORF (arrowhead). A vertical black line shows the multiple cloning site.

    Article Snippet: To construct a TRV expressing ToCV p22 (TRV2p22), the cDNA fragment corresponding to ToCV p22 was PCR amplified using the Expand High Fidelity PCR system (Roche Diagnostics, Mannheim, Germany) and primers MA 1287 and MA 1288 ( ) with specific restriction sites and cloned into pTRV2.

    Techniques: Construct, Plasmid Preparation, Clone Assay, Transmission Assay

    (A) Schematic representation of the VAV-3 cDNAs isolated in this study. Clones obtained from the screening of cDNA libraries are shown in regular typeface. Those obtained from RACE amplifications are shown in italics. The 5′ UTRs are shown as closed boxes. The 3′ UTRs are shown as open boxes and not in scale. The 5′ ends encoding the Vav-3 CH domain are shown as shaded boxes. Stop codons are indicated by asterisks. Start codons are indicated by inverted triangles. Important restriction sites for cloning purposes are indicated. (B) Alignment of the amino acid sequences of the three members of the Vav family in Homo sapiens . Identical residues present in the three proteins are shown on boldface. The individual structural domains are boxed. PRR, proline-rich region.

    Journal: Molecular and Cellular Biology

    Article Title: Biological and Regulatory Properties of Vav-3, a New Member of the Vav Family of Oncoproteins

    doi:

    Figure Lengend Snippet: (A) Schematic representation of the VAV-3 cDNAs isolated in this study. Clones obtained from the screening of cDNA libraries are shown in regular typeface. Those obtained from RACE amplifications are shown in italics. The 5′ UTRs are shown as closed boxes. The 3′ UTRs are shown as open boxes and not in scale. The 5′ ends encoding the Vav-3 CH domain are shown as shaded boxes. Stop codons are indicated by asterisks. Start codons are indicated by inverted triangles. Important restriction sites for cloning purposes are indicated. (B) Alignment of the amino acid sequences of the three members of the Vav family in Homo sapiens . Identical residues present in the three proteins are shown on boldface. The individual structural domains are boxed. PRR, proline-rich region.

    Article Snippet: After obtaining the EST cDNA containing the VAV-3 3′ from the American Tissue Culture Collection, the cDNA fragment was used to screen a human placenta cDNA library (Stratagene).

    Techniques: Isolation, Clone Assay

    Dissection, single-cell isolation, and genome-wide transcriptional profiling of early embryonic mouse heart. Hearts from developing embryos at indicated developmental stages were harvested and dissected into zones as shown. Single cells from each zone underwent lysis, RNA extraction, and cDNA library generation before sequencing and bioinformatics analysis. Workflow for generation and analysis of scRNA-seq data Whole-mount microscopy of murine e8.5, 9.5, and 10.5 heart showing dissection zone boundaries. IFT - inflow tract, A – atrium, V – ventricle, RA – right atrium, LA – left atrium, AVC – atrioventricular canal, RV – right ventricle, RS – right ventricular septum, LS – left ventricular septum, LV – left ventricle, OFT – outflow tract, PO – proximal outflow tract, DO – distal outflow tract. Scale bars = 500 μm. Validation of previously reported chamber-specific genes in dissected e10.5 heart zones by bulk-tissue qPCR. t-SNE plots showing major clusters among all scRNA-seq samples at each stage of development. Color and number labeling indicate zone of origin for each cell. Due to the different number of zones and cells present at each stage, t-SNE plots of different stages should not be directly compared. I – inflow tract. )

    Journal: Developmental cell

    Article Title: Transcriptomic profiling maps anatomically patterned subpopulations among single embryonic cardiac cells

    doi: 10.1016/j.devcel.2016.10.014

    Figure Lengend Snippet: Dissection, single-cell isolation, and genome-wide transcriptional profiling of early embryonic mouse heart. Hearts from developing embryos at indicated developmental stages were harvested and dissected into zones as shown. Single cells from each zone underwent lysis, RNA extraction, and cDNA library generation before sequencing and bioinformatics analysis. Workflow for generation and analysis of scRNA-seq data Whole-mount microscopy of murine e8.5, 9.5, and 10.5 heart showing dissection zone boundaries. IFT - inflow tract, A – atrium, V – ventricle, RA – right atrium, LA – left atrium, AVC – atrioventricular canal, RV – right ventricle, RS – right ventricular septum, LS – left ventricular septum, LV – left ventricle, OFT – outflow tract, PO – proximal outflow tract, DO – distal outflow tract. Scale bars = 500 μm. Validation of previously reported chamber-specific genes in dissected e10.5 heart zones by bulk-tissue qPCR. t-SNE plots showing major clusters among all scRNA-seq samples at each stage of development. Color and number labeling indicate zone of origin for each cell. Due to the different number of zones and cells present at each stage, t-SNE plots of different stages should not be directly compared. I – inflow tract. )

    Article Snippet: To synthesize RNA probes, cDNA fragments (400bp-600bp) of candidate genes were cloned into pBluescript-SK vector (Agilent Technologies) or pGEM-Teasy vector (Promega).

    Techniques: Dissection, Single-cell Isolation, Genome Wide, Lysis, RNA Extraction, cDNA Library Assay, Sequencing, Microscopy, Real-time Polymerase Chain Reaction, Labeling

    CDP/ cut is regulated by PCAF HAT activity through sequences between the C3 and HD and corresponds to the acetylation of CDP/ cut in vivo . ( A ) Schematic diagram depicts various deletion mutants of the CDP/ cut cDNA fused in frame to the DBD of GAL4. The deletion mutants of CDP/ cut cDNA were cloned into parental plasmid pM (CLONTECH) containing the DBD of GAL4. The human CDP/ cut cDNA fragments, fused to GAL4, were tested in a transactivation assay and monitored for the repression of the cotransfected target CAT reporter gene, GAL4InrCAT, as shown in the photo ( Right ). The NULL ( A Upper ) sample corresponds to the background expression of the reporter gene construct GAL4InrCAT. ( B ) Comparison of the relative activities of the individual CDP/ cut cDNAs shown ( A ) fused to the GAL4 DBD is shown in the presence (or absence) of the cotransfected PCAF HAT activity. Results compare the ability of specific CDP/ cut deletions to repress activity of the GAL4InrCAT target. Full-length wild-type CDP/ cut [pG4-CDP (wild type)] and deletion mutant of CDP/ cut (pG4-CDPΔ1186–1256) are compared with the background level of CAT activity rendered in the presence of the parental GAL4 expression vector shown as pGAL4 ( n = 10). ( C ) To analyze the in vivo acetylation of GAL4 derived fusion proteins of CDP/ cut , G4-CDP (wild type) and G4/CDPΔ1186–1256, 8 μg of PCAF (wild type) plasmid was cotransfected with 5 μg of either pG4-CDP (wild type) or pG4/CDPΔ1186–1256 into 293 cells. Acetylation of the CDP/ cut fusions with the DBD of the GAL4 protein was determined by immunoprecipitation of equivalent amounts of G4/CDP (wild type) and G4/CDPΔ1186–1256 protein with a mouse monoclonal anti-GAL4 antibody and precipitated with goat anti-mouse IgG agarose. The immunoprecipitates, separated by SDS/PAGE, were immunoblotted with antiacetylated lysine antibodies ( Upper ). A corresponding immunoblot ( Lower ) by using an anti-GAL4 mAb was performed with the identical cell lysates [as shown ( Upper )] from 293 cells, cotransfected with PCAF and either the parental GAL4 vector pM, pG4-CDPΔ1186–1256, or pG4-CDP (wild type).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of the homeodomain CCAAT displacement/cut protein function by histone acetyltransferases p300/CREB-binding protein (CBP)-associated factor and CBP

    doi:

    Figure Lengend Snippet: CDP/ cut is regulated by PCAF HAT activity through sequences between the C3 and HD and corresponds to the acetylation of CDP/ cut in vivo . ( A ) Schematic diagram depicts various deletion mutants of the CDP/ cut cDNA fused in frame to the DBD of GAL4. The deletion mutants of CDP/ cut cDNA were cloned into parental plasmid pM (CLONTECH) containing the DBD of GAL4. The human CDP/ cut cDNA fragments, fused to GAL4, were tested in a transactivation assay and monitored for the repression of the cotransfected target CAT reporter gene, GAL4InrCAT, as shown in the photo ( Right ). The NULL ( A Upper ) sample corresponds to the background expression of the reporter gene construct GAL4InrCAT. ( B ) Comparison of the relative activities of the individual CDP/ cut cDNAs shown ( A ) fused to the GAL4 DBD is shown in the presence (or absence) of the cotransfected PCAF HAT activity. Results compare the ability of specific CDP/ cut deletions to repress activity of the GAL4InrCAT target. Full-length wild-type CDP/ cut [pG4-CDP (wild type)] and deletion mutant of CDP/ cut (pG4-CDPΔ1186–1256) are compared with the background level of CAT activity rendered in the presence of the parental GAL4 expression vector shown as pGAL4 ( n = 10). ( C ) To analyze the in vivo acetylation of GAL4 derived fusion proteins of CDP/ cut , G4-CDP (wild type) and G4/CDPΔ1186–1256, 8 μg of PCAF (wild type) plasmid was cotransfected with 5 μg of either pG4-CDP (wild type) or pG4/CDPΔ1186–1256 into 293 cells. Acetylation of the CDP/ cut fusions with the DBD of the GAL4 protein was determined by immunoprecipitation of equivalent amounts of G4/CDP (wild type) and G4/CDPΔ1186–1256 protein with a mouse monoclonal anti-GAL4 antibody and precipitated with goat anti-mouse IgG agarose. The immunoprecipitates, separated by SDS/PAGE, were immunoblotted with antiacetylated lysine antibodies ( Upper ). A corresponding immunoblot ( Lower ) by using an anti-GAL4 mAb was performed with the identical cell lysates [as shown ( Upper )] from 293 cells, cotransfected with PCAF and either the parental GAL4 vector pM, pG4-CDPΔ1186–1256, or pG4-CDP (wild type).

    Article Snippet: The cDNA fragments of the human CDP/ cut cDNA were amplified with a Sal I restriction site linker by using the EXpand system (Roche Diagnostics), and corresponding PCR fragments were directly cloned into the EXpand vector II (Roche).

    Techniques: HAT Assay, Activity Assay, In Vivo, Clone Assay, Plasmid Preparation, Transactivation Assay, Expressing, Construct, Mutagenesis, Derivative Assay, Immunoprecipitation, SDS Page