Structured Review

Toyobo cdna
The role of TDP-43 in the expression of VHL, CUL2, and HIF1α. ( a–f ) Measurement of VHL in the presence of overexpression ( a,c,d ) or knock down ( b,e,f ) of TDP-43 in HEK293A cells. ( a,b ) Quantitative real-time PCR analysis of VHL expression in the presence of overexpression or knock down of TDP-43. HEK293A cells were transiently <t>transfected</t> with WT or mutant NLS TDP-43 ( a ) or siRNA oligonucleotides targeting TDP-43 ( b ). At 48 h and 96 h after transfection in ( a ) and ( b ), respectively, cells were harvested for <t>cDNA</t> generation from RNA. In ( b ), plasmid for WT TDP-43 was additionally introduced at 48 h before harvest for the rescue experiment. Each data point represents the average from three ( a ) and six ( b ) cultures (mean ± SD; *p
Cdna, supplied by Toyobo, used in various techniques. Bioz Stars score: 94/100, based on 542 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cdna/product/Toyobo
Average 94 stars, based on 542 article reviews
Price from $9.99 to $1999.99
cdna - by Bioz Stars, 2020-08
94/100 stars

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1) Product Images from "CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS"

Article Title: CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS

Journal: Scientific Reports

doi: 10.1038/srep19118

The role of TDP-43 in the expression of VHL, CUL2, and HIF1α. ( a–f ) Measurement of VHL in the presence of overexpression ( a,c,d ) or knock down ( b,e,f ) of TDP-43 in HEK293A cells. ( a,b ) Quantitative real-time PCR analysis of VHL expression in the presence of overexpression or knock down of TDP-43. HEK293A cells were transiently transfected with WT or mutant NLS TDP-43 ( a ) or siRNA oligonucleotides targeting TDP-43 ( b ). At 48 h and 96 h after transfection in ( a ) and ( b ), respectively, cells were harvested for cDNA generation from RNA. In ( b ), plasmid for WT TDP-43 was additionally introduced at 48 h before harvest for the rescue experiment. Each data point represents the average from three ( a ) and six ( b ) cultures (mean ± SD; *p
Figure Legend Snippet: The role of TDP-43 in the expression of VHL, CUL2, and HIF1α. ( a–f ) Measurement of VHL in the presence of overexpression ( a,c,d ) or knock down ( b,e,f ) of TDP-43 in HEK293A cells. ( a,b ) Quantitative real-time PCR analysis of VHL expression in the presence of overexpression or knock down of TDP-43. HEK293A cells were transiently transfected with WT or mutant NLS TDP-43 ( a ) or siRNA oligonucleotides targeting TDP-43 ( b ). At 48 h and 96 h after transfection in ( a ) and ( b ), respectively, cells were harvested for cDNA generation from RNA. In ( b ), plasmid for WT TDP-43 was additionally introduced at 48 h before harvest for the rescue experiment. Each data point represents the average from three ( a ) and six ( b ) cultures (mean ± SD; *p

Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Plasmid Preparation

2) Product Images from "Lipoamide Acts as an Indirect Antioxidant by Simultaneously Stimulating Mitochondrial Biogenesis and Phase II Antioxidant Enzyme Systems in ARPE-19 Cells"

Article Title: Lipoamide Acts as an Indirect Antioxidant by Simultaneously Stimulating Mitochondrial Biogenesis and Phase II Antioxidant Enzyme Systems in ARPE-19 Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0128502

The effects of LM on oxygen consumption (A), mitochondrial membrane potential (MMP) (B), ROS production (C); cellular ATP level (D) and the expression of MnSOD,Trx2,Prx3,and Prx5 (E). ARPE-19 cells were treated with 40 μmol/L LM for 48 hours; then the following assays were carried out immediately. (A) LM promoted oxygen consumption. Results are expressed as the rate of oxygen consumption, with media without cells used as a blank. Values are means ± SEM from three independent experiments; three parallel measurements were used for each sample in every experiment. (B) LM treatment increased MMP as determined by JC-1 staining. Values are means ± SEM of the ratio of fluorescence at 590 nm to 530 nm from three independent experiments; 4 parallel wells for each group were used in each experiment. (C) LM treatment decreased ROS production examined by DCF-DA staining. Values are means ± SEM of 8 parallel wells of a representative experiment, from four independent experiments each showing similar trends. (D) LM treatment decreased cellular ATP level. Values are means ± SEM from 3 independent experiments. (E) Expression of MnSOD,Trx2,Prx3,and Prx5. ARPE-19 cells were treated with 40 μmol/L LM or LA for 48 h; then RNA was isolated and reverse-transcribed to cDNA. Real time PCR was employed to measure expression levels of the indicated genes. The results (from 5 independent experiments) are expression ratios of the target genes to 18SrRNA, and are normalized to control (control = 100). C stands for control, LM stands for 40 μmol/L LM treatment and LA stands for 40 μmol/L LA treatment. Statistical significance was established by one way ANOVA followed by the Tukey test (A, B, C, D) or LSD test (E). * p
Figure Legend Snippet: The effects of LM on oxygen consumption (A), mitochondrial membrane potential (MMP) (B), ROS production (C); cellular ATP level (D) and the expression of MnSOD,Trx2,Prx3,and Prx5 (E). ARPE-19 cells were treated with 40 μmol/L LM for 48 hours; then the following assays were carried out immediately. (A) LM promoted oxygen consumption. Results are expressed as the rate of oxygen consumption, with media without cells used as a blank. Values are means ± SEM from three independent experiments; three parallel measurements were used for each sample in every experiment. (B) LM treatment increased MMP as determined by JC-1 staining. Values are means ± SEM of the ratio of fluorescence at 590 nm to 530 nm from three independent experiments; 4 parallel wells for each group were used in each experiment. (C) LM treatment decreased ROS production examined by DCF-DA staining. Values are means ± SEM of 8 parallel wells of a representative experiment, from four independent experiments each showing similar trends. (D) LM treatment decreased cellular ATP level. Values are means ± SEM from 3 independent experiments. (E) Expression of MnSOD,Trx2,Prx3,and Prx5. ARPE-19 cells were treated with 40 μmol/L LM or LA for 48 h; then RNA was isolated and reverse-transcribed to cDNA. Real time PCR was employed to measure expression levels of the indicated genes. The results (from 5 independent experiments) are expression ratios of the target genes to 18SrRNA, and are normalized to control (control = 100). C stands for control, LM stands for 40 μmol/L LM treatment and LA stands for 40 μmol/L LA treatment. Statistical significance was established by one way ANOVA followed by the Tukey test (A, B, C, D) or LSD test (E). * p

Techniques Used: Expressing, Staining, Fluorescence, Isolation, Real-time Polymerase Chain Reaction

3) Product Images from "Targeting Gene-Viro-Therapy with AFP driving Apoptin gene shows potent antitumor effect in hepatocarcinoma"

Article Title: Targeting Gene-Viro-Therapy with AFP driving Apoptin gene shows potent antitumor effect in hepatocarcinoma

Journal: Journal of Biomedical Science

doi: 10.1186/1423-0127-19-20

Construction of the dual regulated oncolytic adenovirus AD55-Apoptin and its selective replication in tumor cells . A . Schematic structure of recombinant adenoviruses. Compared with E1B 55KD-deficient adenovirus ZD55, the dual-regulated adenovirus AD55-Apoptin had been further modified with both the E1A promoter replaced by eAFP and the E1b55KD deletion. Then, the Apoptin expression cassette controlled by human cytomegalovirus (HCMV) promoter was obversely inserted to form AD55-Apoptin. ITR is the inverted terminal repeat sequence. B . Detection of E1A levels of recombinant oncolytic adenoviruses. L-02, Huh-7 and PLC was infected with ONYX-015, AD55 and AD55-Apoptin at the MOI of 5, after 48 hours, Western Blotting was conducted to detect E1A protein expression, β-actin was used as a protein loading control. C . 3.5 × 10 5 cells were plated into six-well plates. After 24 h, the cells were infected with 10 MOIs of AD55-Apoptin or AD55 or ONYX-015 or ZD55-EGFP, respectively. After an additional 48 h, medium and cells were scraped into 1.5 ml Eppendorf tube and subjected to three-thaw cycles. The collected supernatant was tested for virus production by standard TCID50 assay on 293 cells. Progeny viruses from 1 MOI of virus were calculated. The results were the average of two independent experiments. D . Huh-7 and QSG-7701 cells were infected with AD55 or AD55-Apoptin at the MOI of 10 for 24, 48 and 96 hours, respectively. The total RNA was collected at the indicated time and reverse transcripted into cDNA, then a real time quantitative PCR was done with the Apoptin or GAPDH primers.
Figure Legend Snippet: Construction of the dual regulated oncolytic adenovirus AD55-Apoptin and its selective replication in tumor cells . A . Schematic structure of recombinant adenoviruses. Compared with E1B 55KD-deficient adenovirus ZD55, the dual-regulated adenovirus AD55-Apoptin had been further modified with both the E1A promoter replaced by eAFP and the E1b55KD deletion. Then, the Apoptin expression cassette controlled by human cytomegalovirus (HCMV) promoter was obversely inserted to form AD55-Apoptin. ITR is the inverted terminal repeat sequence. B . Detection of E1A levels of recombinant oncolytic adenoviruses. L-02, Huh-7 and PLC was infected with ONYX-015, AD55 and AD55-Apoptin at the MOI of 5, after 48 hours, Western Blotting was conducted to detect E1A protein expression, β-actin was used as a protein loading control. C . 3.5 × 10 5 cells were plated into six-well plates. After 24 h, the cells were infected with 10 MOIs of AD55-Apoptin or AD55 or ONYX-015 or ZD55-EGFP, respectively. After an additional 48 h, medium and cells were scraped into 1.5 ml Eppendorf tube and subjected to three-thaw cycles. The collected supernatant was tested for virus production by standard TCID50 assay on 293 cells. Progeny viruses from 1 MOI of virus were calculated. The results were the average of two independent experiments. D . Huh-7 and QSG-7701 cells were infected with AD55 or AD55-Apoptin at the MOI of 10 for 24, 48 and 96 hours, respectively. The total RNA was collected at the indicated time and reverse transcripted into cDNA, then a real time quantitative PCR was done with the Apoptin or GAPDH primers.

Techniques Used: Recombinant, Modification, Expressing, Sequencing, Planar Chromatography, Infection, Western Blot, TCID50 Assay, Real-time Polymerase Chain Reaction

4) Product Images from "Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress"

Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erz384

VIP1 variants that are constitutively localized in the nucleus suppress VIP1–SRDX-dependent root waving. (A, B, and D) Root growth of the transgenic plants expressing GFP, VIP1–GFP, NLS–VIP1–GFP, or GFP–VIP1AAA in the VIP1–SRDX-overexpressing (‘+ VIP1-SRDX’) background. GFP–VIP1AAA was introduced into the VIP1–SRDX-overexpressing background from two individual GFP–VIP1AAA-expressing lines (‘#1’ and ‘#2’), and data for line #2 are presented (see Supplementary Fig. S5 for line #1). These plants were grown for 10 d on agar medium tilted at a 60° angle and photographed to obtain the root vertical growth indices (VGIs). (A) Phenotypes of these 10-day-old seedlings. Scale bar=5 mm. (B) VGIs of these plants. For each box, the top and bottom edges and the middle line indicate the quartiles, and the vertical bar corresponds to the data range ( n =70 for GFP-expressing plants; 40 for VIP1–GFP-expressing plants; 46 for NLS–VIP1–GFP-expressing plants; 29 for GFP–VIP1AAA-expressing plants #2). Data with the same lower case letters are not significantly different ( P > 0.05) according to the Games–Howell test. (C) Expression levels of VIP1-SRDX . Plants were grown as described above, and used for RNA extraction and cDNA synthesis for RT–PCR. Data are means ±SD of three biological replicates. Data with the same lower case letters are not significantly different ( P > 0.05) according to the Tukey–Kramer test. (D) Length of the primary roots of these plants. Data are the same as those used to obtain the VGIs, and are presented as means ±SD. Data with the same lower case letters are not significantly different ( P > 0.05) according to the Games–Howell test.
Figure Legend Snippet: VIP1 variants that are constitutively localized in the nucleus suppress VIP1–SRDX-dependent root waving. (A, B, and D) Root growth of the transgenic plants expressing GFP, VIP1–GFP, NLS–VIP1–GFP, or GFP–VIP1AAA in the VIP1–SRDX-overexpressing (‘+ VIP1-SRDX’) background. GFP–VIP1AAA was introduced into the VIP1–SRDX-overexpressing background from two individual GFP–VIP1AAA-expressing lines (‘#1’ and ‘#2’), and data for line #2 are presented (see Supplementary Fig. S5 for line #1). These plants were grown for 10 d on agar medium tilted at a 60° angle and photographed to obtain the root vertical growth indices (VGIs). (A) Phenotypes of these 10-day-old seedlings. Scale bar=5 mm. (B) VGIs of these plants. For each box, the top and bottom edges and the middle line indicate the quartiles, and the vertical bar corresponds to the data range ( n =70 for GFP-expressing plants; 40 for VIP1–GFP-expressing plants; 46 for NLS–VIP1–GFP-expressing plants; 29 for GFP–VIP1AAA-expressing plants #2). Data with the same lower case letters are not significantly different ( P > 0.05) according to the Games–Howell test. (C) Expression levels of VIP1-SRDX . Plants were grown as described above, and used for RNA extraction and cDNA synthesis for RT–PCR. Data are means ±SD of three biological replicates. Data with the same lower case letters are not significantly different ( P > 0.05) according to the Tukey–Kramer test. (D) Length of the primary roots of these plants. Data are the same as those used to obtain the VGIs, and are presented as means ±SD. Data with the same lower case letters are not significantly different ( P > 0.05) according to the Games–Howell test.

Techniques Used: Transgenic Assay, Expressing, RNA Extraction, Reverse Transcription Polymerase Chain Reaction

Related Articles

Transfection:

Article Title: CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS
Article Snippet: .. Quantitative real-time PCR The total RNA samples were purified from the transfected cells using a commercially available kit (Invitrogen) and were converted to cDNA with reverse transcriptase (Toyobo, Tokyo, Japan). .. The levels of mRNAs for VHL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the synthesized cDNAs were analyzed with the SYBR quantitative PCR kit (Toyobo) and real-time PCR Detection Systems (BIO-RAD).

Synthesized:

Article Title: Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies
Article Snippet: .. Quantification of transcripts (reverse transcriptase–qPCR) Total RNA from Drosophila heads was extracted with TRIzol reagent (Thermo Fisher Scientific, San Jose, CA, USA) and complementary DNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). cDNAs were then analysed by quantitative real-time PCR (BioRad Laboratories, Hercules, CA, USA). ..

Article Title: Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypo-osmotic stress
Article Snippet: .. Total RNA was extracted with a NucleoSpin RNA Plant kit (Macherey-Nagel Düren, Germany). cDNA was synthesized from 500 ng of total RNA with ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan) and oligo(dT)15 primer. ..

Article Title: JunB regulates homeostasis and suppressive functions of effector regulatory T cells
Article Snippet: .. Complementary DNA was synthesized using a Revertra Ace qPCR Kit (Toyobo; FSQ-101) and subjected to qRT-PCR analysis with Faststart SYBR master mix (4673484, Roche) and a Thermal Cycler Dice Real Time system (Takara). .. RNA-sequencing analysis RNA-sequence libraries were prepared with NeoPrep (Illumina) using TruSeq Stranded mRNA NeoPrep Kit (Illumina).

Isolation:

Article Title: Lipoamide Acts as an Indirect Antioxidant by Simultaneously Stimulating Mitochondrial Biogenesis and Phase II Antioxidant Enzyme Systems in ARPE-19 Cells
Article Snippet: .. Total RNA was isolated with Trizol following a standard protocol. cDNA was obtained with Reverse Transcriptase from Toyobo Co. Ltd (Osaka, Japan) and oligo dT primers from Takara Co. (Dalian, China). .. Real-time PCR quantification was preformed using Master SYBR Green Premix and expressed relative to 18S rDNA or 18S rRNA level in the same sample [ ].

Article Title: Scaffold attachment factor B suppresses HIV-1 infection of CD4+ T cells by preventing binding of RNA polymerase II to HIV-1's long terminal repeat
Article Snippet: .. Total cellular RNA from different treated HEK293T cells was isolated by TRIzol reagent (Invitrogen) and reverse-transcribed into complementary DNA using ReverTra Ace qPCR RT Master Mix (Toyobo). .. Total DNA was abstracted with the QIAamp DNA Blood Mini Kit (Qiagen).

Quantitative RT-PCR:

Article Title: A σE-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in Vibrio alginolyticus
Article Snippet: .. Quantitative real-time reverse transcription PCR (qRT-PCR) Equal amounts of RNA (1 μg) were used to generate complementary DNA (Toyobo, Tsuruga, Japan) using random primers. .. Three independent qRT-PCR experiments were performed, and each was run in triplicate.

Article Title: JunB regulates homeostasis and suppressive functions of effector regulatory T cells
Article Snippet: .. Complementary DNA was synthesized using a Revertra Ace qPCR Kit (Toyobo; FSQ-101) and subjected to qRT-PCR analysis with Faststart SYBR master mix (4673484, Roche) and a Thermal Cycler Dice Real Time system (Takara). .. RNA-sequencing analysis RNA-sequence libraries were prepared with NeoPrep (Illumina) using TruSeq Stranded mRNA NeoPrep Kit (Illumina).

Purification:

Article Title: CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS
Article Snippet: .. Quantitative real-time PCR The total RNA samples were purified from the transfected cells using a commercially available kit (Invitrogen) and were converted to cDNA with reverse transcriptase (Toyobo, Tokyo, Japan). .. The levels of mRNAs for VHL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the synthesized cDNAs were analyzed with the SYBR quantitative PCR kit (Toyobo) and real-time PCR Detection Systems (BIO-RAD).

Real-time Polymerase Chain Reaction:

Article Title: Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies
Article Snippet: .. Quantification of transcripts (reverse transcriptase–qPCR) Total RNA from Drosophila heads was extracted with TRIzol reagent (Thermo Fisher Scientific, San Jose, CA, USA) and complementary DNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). cDNAs were then analysed by quantitative real-time PCR (BioRad Laboratories, Hercules, CA, USA). ..

Article Title: CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS
Article Snippet: .. Quantitative real-time PCR The total RNA samples were purified from the transfected cells using a commercially available kit (Invitrogen) and were converted to cDNA with reverse transcriptase (Toyobo, Tokyo, Japan). .. The levels of mRNAs for VHL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the synthesized cDNAs were analyzed with the SYBR quantitative PCR kit (Toyobo) and real-time PCR Detection Systems (BIO-RAD).

Article Title: JunB regulates homeostasis and suppressive functions of effector regulatory T cells
Article Snippet: .. Complementary DNA was synthesized using a Revertra Ace qPCR Kit (Toyobo; FSQ-101) and subjected to qRT-PCR analysis with Faststart SYBR master mix (4673484, Roche) and a Thermal Cycler Dice Real Time system (Takara). .. RNA-sequencing analysis RNA-sequence libraries were prepared with NeoPrep (Illumina) using TruSeq Stranded mRNA NeoPrep Kit (Illumina).

Article Title: Scaffold attachment factor B suppresses HIV-1 infection of CD4+ T cells by preventing binding of RNA polymerase II to HIV-1's long terminal repeat
Article Snippet: .. Total cellular RNA from different treated HEK293T cells was isolated by TRIzol reagent (Invitrogen) and reverse-transcribed into complementary DNA using ReverTra Ace qPCR RT Master Mix (Toyobo). .. Total DNA was abstracted with the QIAamp DNA Blood Mini Kit (Qiagen).

Polymerase Chain Reaction:

Article Title: A σE-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in Vibrio alginolyticus
Article Snippet: .. Quantitative real-time reverse transcription PCR (qRT-PCR) Equal amounts of RNA (1 μg) were used to generate complementary DNA (Toyobo, Tsuruga, Japan) using random primers. .. Three independent qRT-PCR experiments were performed, and each was run in triplicate.

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    Toyobo cdna
    The role of TDP-43 in the expression of VHL, CUL2, and HIF1α. ( a–f ) Measurement of VHL in the presence of overexpression ( a,c,d ) or knock down ( b,e,f ) of TDP-43 in HEK293A cells. ( a,b ) Quantitative real-time PCR analysis of VHL expression in the presence of overexpression or knock down of TDP-43. HEK293A cells were transiently <t>transfected</t> with WT or mutant NLS TDP-43 ( a ) or siRNA oligonucleotides targeting TDP-43 ( b ). At 48 h and 96 h after transfection in ( a ) and ( b ), respectively, cells were harvested for <t>cDNA</t> generation from RNA. In ( b ), plasmid for WT TDP-43 was additionally introduced at 48 h before harvest for the rescue experiment. Each data point represents the average from three ( a ) and six ( b ) cultures (mean ± SD; *p
    Cdna, supplied by Toyobo, used in various techniques. Bioz Stars score: 94/100, based on 861 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdna/product/Toyobo
    Average 94 stars, based on 861 article reviews
    Price from $9.99 to $1999.99
    cdna - by Bioz Stars, 2020-08
    94/100 stars
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    The role of TDP-43 in the expression of VHL, CUL2, and HIF1α. ( a–f ) Measurement of VHL in the presence of overexpression ( a,c,d ) or knock down ( b,e,f ) of TDP-43 in HEK293A cells. ( a,b ) Quantitative real-time PCR analysis of VHL expression in the presence of overexpression or knock down of TDP-43. HEK293A cells were transiently transfected with WT or mutant NLS TDP-43 ( a ) or siRNA oligonucleotides targeting TDP-43 ( b ). At 48 h and 96 h after transfection in ( a ) and ( b ), respectively, cells were harvested for cDNA generation from RNA. In ( b ), plasmid for WT TDP-43 was additionally introduced at 48 h before harvest for the rescue experiment. Each data point represents the average from three ( a ) and six ( b ) cultures (mean ± SD; *p

    Journal: Scientific Reports

    Article Title: CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS

    doi: 10.1038/srep19118

    Figure Lengend Snippet: The role of TDP-43 in the expression of VHL, CUL2, and HIF1α. ( a–f ) Measurement of VHL in the presence of overexpression ( a,c,d ) or knock down ( b,e,f ) of TDP-43 in HEK293A cells. ( a,b ) Quantitative real-time PCR analysis of VHL expression in the presence of overexpression or knock down of TDP-43. HEK293A cells were transiently transfected with WT or mutant NLS TDP-43 ( a ) or siRNA oligonucleotides targeting TDP-43 ( b ). At 48 h and 96 h after transfection in ( a ) and ( b ), respectively, cells were harvested for cDNA generation from RNA. In ( b ), plasmid for WT TDP-43 was additionally introduced at 48 h before harvest for the rescue experiment. Each data point represents the average from three ( a ) and six ( b ) cultures (mean ± SD; *p

    Article Snippet: Quantitative real-time PCR The total RNA samples were purified from the transfected cells using a commercially available kit (Invitrogen) and were converted to cDNA with reverse transcriptase (Toyobo, Tokyo, Japan).

    Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Plasmid Preparation

    Site and frequency of transcriptional slippage at the ClYVV P3 region in planta . ( a ) Indel frequencies were estimated by a negative binomial regression using indel counts observed by amplicon sequencing of the P3 region encompassing the G 2 A 6 motif. Amplicons prepared from cDNA of systemically propagated Cl30 or RB virus (solid line), or from their parental plasmids used for inoculation (pCl30 or pRB; dashed line), were subjected to sequencing. Estimated frequencies of each size of indels are shown. Error bars indicate standard errors of the estimated frequencies. Asterisks indicate an indel size in which the viral RNA showed a higher frequency than the corresponding control plasmid ( p

    Journal: Scientific Reports

    Article Title: Truncated yet functional viral protein produced via RNA polymerase slippage implies underestimated coding capacity of RNA viruses

    doi: 10.1038/srep21411

    Figure Lengend Snippet: Site and frequency of transcriptional slippage at the ClYVV P3 region in planta . ( a ) Indel frequencies were estimated by a negative binomial regression using indel counts observed by amplicon sequencing of the P3 region encompassing the G 2 A 6 motif. Amplicons prepared from cDNA of systemically propagated Cl30 or RB virus (solid line), or from their parental plasmids used for inoculation (pCl30 or pRB; dashed line), were subjected to sequencing. Estimated frequencies of each size of indels are shown. Error bars indicate standard errors of the estimated frequencies. Asterisks indicate an indel size in which the viral RNA showed a higher frequency than the corresponding control plasmid ( p

    Article Snippet: The 209-nt amplicons were prepared in 50 μl reaction mixtures containing 1 μl of cDNA solutions, 25 μl 2 × PCR buffer for KOD-FX neo (TOYOBO), 0.4 mM dNTPs, 1 U KOD-FX neo (TOYOBO) and 0.3 μM primer pairs: 3879 and 3881 for samples of Cl30; and 3880 and 3882 for samples of RB ( ).

    Techniques: Amplification, Sequencing, Plasmid Preparation

    Recovery of recombinant SYNV in N . benthamiana . (A) Schematic representation of the SYNV infectious cDNA clone and supporting plasmids. The pSYNV plasmid is designed for transcription to yield the SYNV antigenome (ag) RNA and contains the full-length SYNV cDNA positioned between a truncated CaMV double 35S promoter (2X35S) and the HDV ribozyme sequence in the pCB301 plasmid. Note that the SYNV gene order is shown in the antigenome sense. The pGD-N, pGD-P and pGD-L supporting plasmids encode the viral N, P and L core NC proteins, respectively, and the pGD-VSRs encode the TBSV p19, BSMV γB, and TEV P1/HC-Pro suppressors of RNA silencing (VSRs). le: leader; tr: trailer; Rz: ribozyme; LB: left border sequence; RB: right border sequence; Nos: nopaline synthase terminator. (B) Symptoms of N . benthamiana plants systemically infected with rSYNV. Agrobacterium cultures containing the pSYNV, pGD-N, pGD-P, pGD-L and the three VSRs plasmids were mixed and infiltrated into N . benthamiana leaves. Infected plants showing stunting and typical vein clearing symptoms were photographed at 35 days post infiltration (dpi) along with a mock control (uninfected healthy plant). (C) Detection of viral structural proteins in wtSYNV- and rSYNV-infected N . benthamiana plants. Total protein samples were analyzed by Western blotting with anti-SYNV virion antibody. The Coomassie blue stained Rubisco large subunit (Rub L) was used as a loading control. The positions of the SYNV G, N, M and P proteins are indicated along the left side of the gel and the numbers along the right side are protein size markers in KDa. (D) Restriction enzyme site identification of wtSYNV and rSYNV cDNAs. Total RNAs were extracted from wtSYNV- and rSYNV-infected plants and used as templates for reverse transcription PCR (RT-PCR). The wtSYNV and rSYNV RT-PCR products (∼1500 bp) were digested with Bsm BI or Apa I and analyzed on 1.5% agarose gels. Positions of DNA size markers (M) are shown in base pairs.

    Journal: PLoS Pathogens

    Article Title: Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

    doi: 10.1371/journal.ppat.1005223

    Figure Lengend Snippet: Recovery of recombinant SYNV in N . benthamiana . (A) Schematic representation of the SYNV infectious cDNA clone and supporting plasmids. The pSYNV plasmid is designed for transcription to yield the SYNV antigenome (ag) RNA and contains the full-length SYNV cDNA positioned between a truncated CaMV double 35S promoter (2X35S) and the HDV ribozyme sequence in the pCB301 plasmid. Note that the SYNV gene order is shown in the antigenome sense. The pGD-N, pGD-P and pGD-L supporting plasmids encode the viral N, P and L core NC proteins, respectively, and the pGD-VSRs encode the TBSV p19, BSMV γB, and TEV P1/HC-Pro suppressors of RNA silencing (VSRs). le: leader; tr: trailer; Rz: ribozyme; LB: left border sequence; RB: right border sequence; Nos: nopaline synthase terminator. (B) Symptoms of N . benthamiana plants systemically infected with rSYNV. Agrobacterium cultures containing the pSYNV, pGD-N, pGD-P, pGD-L and the three VSRs plasmids were mixed and infiltrated into N . benthamiana leaves. Infected plants showing stunting and typical vein clearing symptoms were photographed at 35 days post infiltration (dpi) along with a mock control (uninfected healthy plant). (C) Detection of viral structural proteins in wtSYNV- and rSYNV-infected N . benthamiana plants. Total protein samples were analyzed by Western blotting with anti-SYNV virion antibody. The Coomassie blue stained Rubisco large subunit (Rub L) was used as a loading control. The positions of the SYNV G, N, M and P proteins are indicated along the left side of the gel and the numbers along the right side are protein size markers in KDa. (D) Restriction enzyme site identification of wtSYNV and rSYNV cDNAs. Total RNAs were extracted from wtSYNV- and rSYNV-infected plants and used as templates for reverse transcription PCR (RT-PCR). The wtSYNV and rSYNV RT-PCR products (∼1500 bp) were digested with Bsm BI or Apa I and analyzed on 1.5% agarose gels. Positions of DNA size markers (M) are shown in base pairs.

    Article Snippet: Construction of SYNV infectious clones Total RNA extracted from SYNV-infected N . benthamiana plants was used for RT-PCR amplification of the full-length cDNA of agRNA with a high-fidelity KOD-Plus-Neo DNA polymerase (Toyobo, Osaka, Japan) and the forward 5′-tttcatttggagaggAGAGACAGAAACTCAGAAAATACAAT-3′ and reverse 5′-atgccatgccgacccAGAGACAAAAGCTCAGAACAATCCCTAT-3′ primers.

    Techniques: Recombinant, Plasmid Preparation, Sequencing, Infection, Western Blot, Staining, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Targeted disruption of the mouse Fbx15 gene. (A) Structures of the Fbx15 genomic locus, a targeting vector, and the targeted locus generated by homologous recombination. The targeting vector contains the β- geo cassette in place of exons 3 to 7. The length of the diagnostic Hin dIII (H) restriction fragments and the locations of the 5′ and 3′ probes for Southern blot analyses are shown. Arrows indicate the primers for PCR analysis. The diagrams are not drawn to scale. (B) Southern blot analysis. Specific hybridization with the 5′ probe produces a 10-kb band from the wild-type locus and an 8-kb band from the targeted locus. Hybridization with the 3′ probe produces a 9-kb band from the wild-type locus and a 10-kb band from the targeted locus. +/+ and ± represent genotypes of Fbx15 +/+ and Fbx15 +/− cells, respectively. (C) PCR analysis. PCR with the three primers shown in panel A produces a 280-bp band form the wild-type locus and a 725-bp band from the targeted locus. (D) Northern blot analysis. Total RNA isolated from ES cells of each genotype was separated, blotted, and hybridized to cDNA probes of Fbx15 and Oct3/4. (E) Western blot analyses. Lysates isolated from ES cells of each genotype were separated by SDS-PAGE and immunoblotted with anti-Fbx15 serum or anti-CDK4 antibody.

    Journal: Molecular and Cellular Biology

    Article Title: Fbx15 Is a Novel Target of Oct3/4 but Is Dispensable for Embryonic Stem Cell Self-Renewal and Mouse Development

    doi: 10.1128/MCB.23.8.2699-2708.2003

    Figure Lengend Snippet: Targeted disruption of the mouse Fbx15 gene. (A) Structures of the Fbx15 genomic locus, a targeting vector, and the targeted locus generated by homologous recombination. The targeting vector contains the β- geo cassette in place of exons 3 to 7. The length of the diagnostic Hin dIII (H) restriction fragments and the locations of the 5′ and 3′ probes for Southern blot analyses are shown. Arrows indicate the primers for PCR analysis. The diagrams are not drawn to scale. (B) Southern blot analysis. Specific hybridization with the 5′ probe produces a 10-kb band from the wild-type locus and an 8-kb band from the targeted locus. Hybridization with the 3′ probe produces a 9-kb band from the wild-type locus and a 10-kb band from the targeted locus. +/+ and ± represent genotypes of Fbx15 +/+ and Fbx15 +/− cells, respectively. (C) PCR analysis. PCR with the three primers shown in panel A produces a 280-bp band form the wild-type locus and a 725-bp band from the targeted locus. (D) Northern blot analysis. Total RNA isolated from ES cells of each genotype was separated, blotted, and hybridized to cDNA probes of Fbx15 and Oct3/4. (E) Western blot analyses. Lysates isolated from ES cells of each genotype were separated by SDS-PAGE and immunoblotted with anti-Fbx15 serum or anti-CDK4 antibody.

    Article Snippet: First-strand cDNA was synthesized from total RNA with ReverTra Ace-α (Toyobo).

    Techniques: Plasmid Preparation, Generated, Homologous Recombination, Diagnostic Assay, Southern Blot, Polymerase Chain Reaction, Hybridization, Northern Blot, Isolation, Western Blot, SDS Page