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    Thermo Fisher superscript iii ssiii first strand synthesis system
    Induction of the GSR system under starvation. B . abortus 2308 wt (black bars) and the isogenic lovhk :: km (dark grey bars) and ∆lovR (light grey bars) mutant strains were grown in TSB medium up to logarithmic phase. First, an aliquot was drawn (time 0 h), then the rest of the culture was washed and resuspended in modified MM1 minimal medium, and aliquots were drawn at 0.5 h, 1 h and 2 h. Expression of phyR gene was analyzed by <t>qRT-PCR.</t> The if-1 housekeeping gene was used as a reference. Data represent the average of <t>three</t> independent experiments and are reported as fold induction relative to wt at time 0 h in TSB ± standard error. p -values between each strain and wt (at each time point) were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p
    Superscript Iii Ssiii First Strand Synthesis System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher superscript iii first strand synthesis system thermo fisher scientific
    Induction of the GSR system under starvation. B . abortus 2308 wt (black bars) and the isogenic lovhk :: km (dark grey bars) and ∆lovR (light grey bars) mutant strains were grown in TSB medium up to logarithmic phase. First, an aliquot was drawn (time 0 h), then the rest of the culture was washed and resuspended in modified MM1 minimal medium, and aliquots were drawn at 0.5 h, 1 h and 2 h. Expression of phyR gene was analyzed by <t>qRT-PCR.</t> The if-1 housekeeping gene was used as a reference. Data represent the average of <t>three</t> independent experiments and are reported as fold induction relative to wt at time 0 h in TSB ± standard error. p -values between each strain and wt (at each time point) were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p
    Superscript Iii First Strand Synthesis System Thermo Fisher Scientific, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii first strand synthesis system thermo fisher scientific/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superscript iii first strand synthesis system thermo fisher scientific - by Bioz Stars, 2020-08
    91/100 stars
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    92
    Fisher Scientific superscript iii first strand synthesis system thermo fisher scientific
    Induction of the GSR system under starvation. B . abortus 2308 wt (black bars) and the isogenic lovhk :: km (dark grey bars) and ∆lovR (light grey bars) mutant strains were grown in TSB medium up to logarithmic phase. First, an aliquot was drawn (time 0 h), then the rest of the culture was washed and resuspended in modified MM1 minimal medium, and aliquots were drawn at 0.5 h, 1 h and 2 h. Expression of phyR gene was analyzed by <t>qRT-PCR.</t> The if-1 housekeeping gene was used as a reference. Data represent the average of <t>three</t> independent experiments and are reported as fold induction relative to wt at time 0 h in TSB ± standard error. p -values between each strain and wt (at each time point) were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p
    Superscript Iii First Strand Synthesis System Thermo Fisher Scientific, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii first strand synthesis system thermo fisher scientific/product/Fisher Scientific
    Average 92 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    superscript iii first strand synthesis system thermo fisher scientific - by Bioz Stars, 2020-08
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    99
    Thermo Fisher superscript iii first strand synthesis ssiii supermix
    STIM1 depletion reduces ER Ca 2+ content in T cells. ( A ) Expression of STIM1 ( Left ) and Orai1 ( Right ) in control and JP4-depleted Jurkat cells detected by immunoblotting and quantitative <t>RT-PCR,</t> respectively. The transcript data show mean ± SEM of triplicates. ( B ) STIM1 expression in control (Scr) or STIM1-depleted (STIM1 KD) Jurkat cells determined by immunoblotting. β-Actin was used as a loading control. ( C ) SOCE and ER Ca 2+ content measurements in control (Scr) or STIM1-depleted (STIM1 KD) Jurkat cells after passive store depletion with thapsigargin (TG) (1 μM) in Ca 2+ -free solution, and addition of 2 mM Ca 2+ -containing solution ( Left ). Traces show averaged (±SEM) responses from 30 to 50 Jurkat T cells. Bar graphs show change in ER Ca 2+ content and SOCE (±SEM) from <t>three</t> independent experiments. Control (Scr) and STIM1-depleted (KD) Jurkat cells were treated with ionomycin (1 μM, iono) in Ca 2+ -free solution to measure the ER Ca 2+ content ( Right two panels). Traces show averaged (±SEM) responses from 30 to 50 cells, and bar graph shows change in ER Ca 2+ content (±SEM) from three independent experiments. * P
    Superscript Iii First Strand Synthesis Ssiii Supermix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Induction of the GSR system under starvation. B . abortus 2308 wt (black bars) and the isogenic lovhk :: km (dark grey bars) and ∆lovR (light grey bars) mutant strains were grown in TSB medium up to logarithmic phase. First, an aliquot was drawn (time 0 h), then the rest of the culture was washed and resuspended in modified MM1 minimal medium, and aliquots were drawn at 0.5 h, 1 h and 2 h. Expression of phyR gene was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments and are reported as fold induction relative to wt at time 0 h in TSB ± standard error. p -values between each strain and wt (at each time point) were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p

    Journal: PLoS ONE

    Article Title: LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

    doi: 10.1371/journal.pone.0124058

    Figure Lengend Snippet: Induction of the GSR system under starvation. B . abortus 2308 wt (black bars) and the isogenic lovhk :: km (dark grey bars) and ∆lovR (light grey bars) mutant strains were grown in TSB medium up to logarithmic phase. First, an aliquot was drawn (time 0 h), then the rest of the culture was washed and resuspended in modified MM1 minimal medium, and aliquots were drawn at 0.5 h, 1 h and 2 h. Expression of phyR gene was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments and are reported as fold induction relative to wt at time 0 h in TSB ± standard error. p -values between each strain and wt (at each time point) were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p

    Article Snippet: Real Time quantitative RT-PCR assays Reverse transcription was performed with SuperScript III First Strand Synthesis System (Invitrogen) following the manufacturer’s instructions using random decamer primers (Invitrogen) and RNasin ribonuclease inhibitor (Promega).

    Techniques: Mutagenesis, Modification, Expressing, Quantitative RT-PCR

    LOVHK modulates the GSR system in B . abortus . B . abortus 2308 wt and in the isogenic lovhk :: km , ∆lovR , ∆phyR and lovhk :: km /pMR_ lovhk strains were cultured in TSB up to logarithmic phase, and expression of lovhk , lovR , dps and rpoH1 genes was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments, and are reported as fold induction relative to wt ± standard error. p -values between each strain and wt were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (** = p

    Journal: PLoS ONE

    Article Title: LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

    doi: 10.1371/journal.pone.0124058

    Figure Lengend Snippet: LOVHK modulates the GSR system in B . abortus . B . abortus 2308 wt and in the isogenic lovhk :: km , ∆lovR , ∆phyR and lovhk :: km /pMR_ lovhk strains were cultured in TSB up to logarithmic phase, and expression of lovhk , lovR , dps and rpoH1 genes was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments, and are reported as fold induction relative to wt ± standard error. p -values between each strain and wt were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (** = p

    Article Snippet: Real Time quantitative RT-PCR assays Reverse transcription was performed with SuperScript III First Strand Synthesis System (Invitrogen) following the manufacturer’s instructions using random decamer primers (Invitrogen) and RNasin ribonuclease inhibitor (Promega).

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR

    PhyR expression is decreased in the lovhk mutant. A. B . abortus 2308 wt and the isogenic lovhk :: km , ∆lovR and lovhk :: km /pMR_ lovhk strains were cultured in TSB up to logarithmic phase, and expression of the phyR gene was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments, and are reported as fold induction relative to wt ± standard error. p -values between each strain and wt were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p

    Journal: PLoS ONE

    Article Title: LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

    doi: 10.1371/journal.pone.0124058

    Figure Lengend Snippet: PhyR expression is decreased in the lovhk mutant. A. B . abortus 2308 wt and the isogenic lovhk :: km , ∆lovR and lovhk :: km /pMR_ lovhk strains were cultured in TSB up to logarithmic phase, and expression of the phyR gene was analyzed by qRT-PCR. The if-1 housekeeping gene was used as a reference. Data represent the average of three independent experiments, and are reported as fold induction relative to wt ± standard error. p -values between each strain and wt were determined by one-way ANOVA and post-hoc Tukey’s multiple comparisons test (* = p

    Article Snippet: Real Time quantitative RT-PCR assays Reverse transcription was performed with SuperScript III First Strand Synthesis System (Invitrogen) following the manufacturer’s instructions using random decamer primers (Invitrogen) and RNasin ribonuclease inhibitor (Promega).

    Techniques: Expressing, Mutagenesis, Cell Culture, Quantitative RT-PCR

    The effect of SNHG20 knockdown on cell growth, apoptosis, and colony formation ( A ) The mRNA level of SNHG20 in ovarian cancer cells, which were transfected with siSNHG20, was detected, siNC acts as normal control. MTT assays showed SNHG20 knockdown inhibited cell proliferation of SKOV3 ( B ), OVCA429 ( C ), and OVCA433 ( D ) cells. ( E ) Flow cytometry analysis was performed for testing the effect of SNHG20 knockdown on the apoptosis of ovarian cancer cells. ( F ) Colony formation assay measuring colony formation of SNHG20 knockdown ovarian cancer cells. SNHG20 knockdown ovarian cancer cells were inoculated into nude mice and the tumor volume was detected every week. The growth curve of SKOV3 ( G ), OVCA429 ( H ), and OVCA433 ( I ) tumors were determined every week. Colony number was normalized to that obtained with cells transfected with siNC, which was set to 100%. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Journal: Bioscience Reports

    Article Title: Up-regulation of long non-coding RNA SNHG20 promotes ovarian cancer progression via Wnt/β-catenin signaling

    doi: 10.1042/BSR20170681

    Figure Lengend Snippet: The effect of SNHG20 knockdown on cell growth, apoptosis, and colony formation ( A ) The mRNA level of SNHG20 in ovarian cancer cells, which were transfected with siSNHG20, was detected, siNC acts as normal control. MTT assays showed SNHG20 knockdown inhibited cell proliferation of SKOV3 ( B ), OVCA429 ( C ), and OVCA433 ( D ) cells. ( E ) Flow cytometry analysis was performed for testing the effect of SNHG20 knockdown on the apoptosis of ovarian cancer cells. ( F ) Colony formation assay measuring colony formation of SNHG20 knockdown ovarian cancer cells. SNHG20 knockdown ovarian cancer cells were inoculated into nude mice and the tumor volume was detected every week. The growth curve of SKOV3 ( G ), OVCA429 ( H ), and OVCA433 ( I ) tumors were determined every week. Colony number was normalized to that obtained with cells transfected with siNC, which was set to 100%. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Article Snippet: The total RNA was reverse transcribed into first-strand cDNA using the SuperScript III® (Invitrogen) according to the manufacturer’s guide. q-PCR was performed on the SYBR Premix ExTaq kit (TaKaRa) on ABIPRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, FosterCity, CA, U.S.A.) according to the instructions.

    Techniques: Transfection, MTT Assay, Flow Cytometry, Cytometry, Colony Assay, Mouse Assay, Quantitative RT-PCR

    LncRNA SNHG20 expression was increased in ovarian cancer ( A ) The expression of SNHG20 in 17 ovarian tumor samples and paired adjacent non-tumorous normal tissues was examined by q-PCR. ( B ) The expression in 13 metastatic ovarian tumor samples and paired primary tumor samples was also examined by q-PCR. ( C ) The expression of SNHG20 in HOSE and ovarian cancer cells was determined by q-PCR. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Journal: Bioscience Reports

    Article Title: Up-regulation of long non-coding RNA SNHG20 promotes ovarian cancer progression via Wnt/β-catenin signaling

    doi: 10.1042/BSR20170681

    Figure Lengend Snippet: LncRNA SNHG20 expression was increased in ovarian cancer ( A ) The expression of SNHG20 in 17 ovarian tumor samples and paired adjacent non-tumorous normal tissues was examined by q-PCR. ( B ) The expression in 13 metastatic ovarian tumor samples and paired primary tumor samples was also examined by q-PCR. ( C ) The expression of SNHG20 in HOSE and ovarian cancer cells was determined by q-PCR. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Article Snippet: The total RNA was reverse transcribed into first-strand cDNA using the SuperScript III® (Invitrogen) according to the manufacturer’s guide. q-PCR was performed on the SYBR Premix ExTaq kit (TaKaRa) on ABIPRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, FosterCity, CA, U.S.A.) according to the instructions.

    Techniques: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR

    The effect of SNHG20 overexpression on normal ovarian epithelial cell growth, apoptosis, and colony formation ( A ) HOSE cells were infected with lentivirus carrying SNHG20 and then the mRNA level was examined by q-PCR. MTT assays showed the effects of SNHG20 overexpression on HOSE cell growth ( B ), cell apoptosis ( C ), and colony formation ( D ). Colony number was normalized to that obtained from cells transfected with siNC, which was set to 100%. ( E ) SNHG20 overexpressing HOSE cells were inoculated into nude mice and the tumor volumes were evaluated every week. ( F ) The tumor weight was detected at the end point after we killed all groups of mice. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Journal: Bioscience Reports

    Article Title: Up-regulation of long non-coding RNA SNHG20 promotes ovarian cancer progression via Wnt/β-catenin signaling

    doi: 10.1042/BSR20170681

    Figure Lengend Snippet: The effect of SNHG20 overexpression on normal ovarian epithelial cell growth, apoptosis, and colony formation ( A ) HOSE cells were infected with lentivirus carrying SNHG20 and then the mRNA level was examined by q-PCR. MTT assays showed the effects of SNHG20 overexpression on HOSE cell growth ( B ), cell apoptosis ( C ), and colony formation ( D ). Colony number was normalized to that obtained from cells transfected with siNC, which was set to 100%. ( E ) SNHG20 overexpressing HOSE cells were inoculated into nude mice and the tumor volumes were evaluated every week. ( F ) The tumor weight was detected at the end point after we killed all groups of mice. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Article Snippet: The total RNA was reverse transcribed into first-strand cDNA using the SuperScript III® (Invitrogen) according to the manufacturer’s guide. q-PCR was performed on the SYBR Premix ExTaq kit (TaKaRa) on ABIPRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, FosterCity, CA, U.S.A.) according to the instructions.

    Techniques: Over Expression, Infection, Polymerase Chain Reaction, MTT Assay, Transfection, Mouse Assay, Quantitative RT-PCR

    The effect of SNHG20 knockdown on Wnt/β-catenin signaling pathway in ovarian cancer cells Ovarian cancer cells were transfected with siSNHG20 or siNC, then these cells were subjected to q-PCR ( A ) and Western blot ( B ) for testing the mRNA and protein level of E-cadherin. ( C–E ) The mRNA level and protein level of β-catenin, GSK-3β, p-GSK-3β and several targets of Wnt/β-catenin signaling were examined by q-PCR and western blot in SKOV3 and OVCA429 cells. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Journal: Bioscience Reports

    Article Title: Up-regulation of long non-coding RNA SNHG20 promotes ovarian cancer progression via Wnt/β-catenin signaling

    doi: 10.1042/BSR20170681

    Figure Lengend Snippet: The effect of SNHG20 knockdown on Wnt/β-catenin signaling pathway in ovarian cancer cells Ovarian cancer cells were transfected with siSNHG20 or siNC, then these cells were subjected to q-PCR ( A ) and Western blot ( B ) for testing the mRNA and protein level of E-cadherin. ( C–E ) The mRNA level and protein level of β-catenin, GSK-3β, p-GSK-3β and several targets of Wnt/β-catenin signaling were examined by q-PCR and western blot in SKOV3 and OVCA429 cells. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Article Snippet: The total RNA was reverse transcribed into first-strand cDNA using the SuperScript III® (Invitrogen) according to the manufacturer’s guide. q-PCR was performed on the SYBR Premix ExTaq kit (TaKaRa) on ABIPRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, FosterCity, CA, U.S.A.) according to the instructions.

    Techniques: Transfection, Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR

    Expression variations between SG and SYN obtained using NormFinder software over the blood-feeding duration. The data are the average of three biological replications. The circles are for intergroup variation and error bars are for intragroup variation

    Journal: Journal of medical entomology

    Article Title: Validation of Internal Reference Genes for Real-Time Quantitative Polymerase Chain Reaction Studies in the Tick, Ixodes scapularis (Acari: Ixodidae)

    doi:

    Figure Lengend Snippet: Expression variations between SG and SYN obtained using NormFinder software over the blood-feeding duration. The data are the average of three biological replications. The circles are for intergroup variation and error bars are for intragroup variation

    Article Snippet: Total RNA, 340 ng from SG and 40–60 ng from SYN, was used to synthesize first-strand cDNA using the SuperScript III system (Invitrogen, Life sciences, Grand Island, NY)and an oligo-dT-based reverse transcriptase protocol.

    Techniques: Expressing, Software

    Detection of processed transcripts of E. coli CRISPR I cassette A. The E. coli CRISPR I locus is schematically shown. Genes are indicated by arrows and identified. In CRISPR I cassette, repeats are indicated by black rhombuses, spacers – by white rectangles. Numbers below the scheme indicate spacer numbers. The last repeat of the cassette is colored grey, to indicate that its sequence differs from other repeats. The leader sequence is located between the CRISPR I cassette and the cas2 gene and is indicated by a thicker black line. B. A Northern blot showing changes in abundance of spacer 4 transcript in E. coli strains with indicated cas genes disruptions. The probe was designed to reveal RNA produced by rightward (see Fig. 1A) transcription. C. Northern blots with total RNA purified from E. coli cells with disrupted casA gene were performed with probes specific for spacers indicated (direction of transcription the same as in Fig. 1B). In each panel, the first three lanes contained increasing amounts of DNA oligonucleotides complementary to probes used for blotting. Approximate calculated numbers of processed CRISPR transcript per cell are shown below.

    Journal: Molecular microbiology

    Article Title: Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli

    doi: 10.1111/j.1365-2958.2010.07265.x

    Figure Lengend Snippet: Detection of processed transcripts of E. coli CRISPR I cassette A. The E. coli CRISPR I locus is schematically shown. Genes are indicated by arrows and identified. In CRISPR I cassette, repeats are indicated by black rhombuses, spacers – by white rectangles. Numbers below the scheme indicate spacer numbers. The last repeat of the cassette is colored grey, to indicate that its sequence differs from other repeats. The leader sequence is located between the CRISPR I cassette and the cas2 gene and is indicated by a thicker black line. B. A Northern blot showing changes in abundance of spacer 4 transcript in E. coli strains with indicated cas genes disruptions. The probe was designed to reveal RNA produced by rightward (see Fig. 1A) transcription. C. Northern blots with total RNA purified from E. coli cells with disrupted casA gene were performed with probes specific for spacers indicated (direction of transcription the same as in Fig. 1B). In each panel, the first three lanes contained increasing amounts of DNA oligonucleotides complementary to probes used for blotting. Approximate calculated numbers of processed CRISPR transcript per cell are shown below.

    Article Snippet: For a single extension reaction up to 0.5 µg of in vitro transcribed RNA was reverse-transcribed with 100 U of SuperScript III enzyme of First-Strand Synthesis Kit for RT-PCR (Invitrogen) according to the manufacturer’s protocol in the presence of 1 pmoles of [32 P] 5’-end-labelled primers.

    Techniques: CRISPR, Sequencing, Northern Blot, Produced, Purification

    HbCIPK2 interacted with HbFd1. (A) Examination of the interaction between HbCIPK2 and HbFd1 in yeast. The interactions were verified by the yeast growing on selective medium (SC/-Leu-Trp-His-Ura with 35 mM 3-AT) and conducting ß-Gal assays. Bait vector with HbCIPK2 or prey vector with HbFd1 was transformed with empty vector as negative control, and the plasmids pPC97-Fos and pPC86-Jun provided by Invitrogen system were used as positive control. (B) Co-BiFC assay to verify the interaction of HbCIPK2 and HbFd1 in Arabidopsis protoplasts. Construct AtCBF1::RFP was used as reference to co-localize the interaction of HbFd1 with HbCIPK2, and transformants expressing SPYNE/HbFd1::SPYCE and SPYNE::HbCIPK2/SPYCE were used as negative controls. (C) Co-IP assay to show the interaction between HbCIPK2 and mature HbFd1 in cell line HEK293, co-expressing HbCIPK2-Myc and HbFd1△cTP-Flag. Cell proteins before (Input) and after (IP) immunoprecipitation were separated in SDS-PAGE gels, transferred onto the nitrocellulose membranes, and analyzed by protein gel blotting with antibodies as indicated. All assays repeated three times.

    Journal: PLoS ONE

    Article Title: Identification and Expression Analysis of a Novel HbCIPK2-Interacting Ferredoxin from Halophyte H. brevisubulatum

    doi: 10.1371/journal.pone.0144132

    Figure Lengend Snippet: HbCIPK2 interacted with HbFd1. (A) Examination of the interaction between HbCIPK2 and HbFd1 in yeast. The interactions were verified by the yeast growing on selective medium (SC/-Leu-Trp-His-Ura with 35 mM 3-AT) and conducting ß-Gal assays. Bait vector with HbCIPK2 or prey vector with HbFd1 was transformed with empty vector as negative control, and the plasmids pPC97-Fos and pPC86-Jun provided by Invitrogen system were used as positive control. (B) Co-BiFC assay to verify the interaction of HbCIPK2 and HbFd1 in Arabidopsis protoplasts. Construct AtCBF1::RFP was used as reference to co-localize the interaction of HbFd1 with HbCIPK2, and transformants expressing SPYNE/HbFd1::SPYCE and SPYNE::HbCIPK2/SPYCE were used as negative controls. (C) Co-IP assay to show the interaction between HbCIPK2 and mature HbFd1 in cell line HEK293, co-expressing HbCIPK2-Myc and HbFd1△cTP-Flag. Cell proteins before (Input) and after (IP) immunoprecipitation were separated in SDS-PAGE gels, transferred onto the nitrocellulose membranes, and analyzed by protein gel blotting with antibodies as indicated. All assays repeated three times.

    Article Snippet: Total RNA from H . brevisubulatum seedling treated with various stresses (350 mM NaCl, 350 mM mannitol and 10% PEG6000 stressed for 6 hrs, and 4°C for 12 hrs, respectively) upon the emergence of the third shoot was extracted using RNeasy Plant Mini kit (QIAGEN, Stockach, Germany) and first strand cDNA was synthesized using the SuperScript III First-Strand (Invitrogen).

    Techniques: Plasmid Preparation, Transformation Assay, Negative Control, Positive Control, Bimolecular Fluorescence Complementation Assay, Construct, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page

    Ag-specific CD4 + T cells are not recruited to the infected kidney. Leukocytes from naive and infected kidneys ( A ) or small intestine ( B ) of mice, treated as in Fig. 1 , were analyzed by flow cytometry for the presence of OT.II (Vα2 + CD45.1 + ) cells. Example plots are gated on CD4 + lymphocytes and are representative of ≥12 animals from four experiments. Graph in (A) shows the frequency of OT.II cells in the kidney in naive ( n = 4) and infected ( n = 15) mice at day 3 postinfection. ( C ) Frequency of OT.I (Vα2 + CD45.1 + ) cells in naive ( n = 2) and infected ( n = 10) kidneys at day 3 postinfection. Example plot is representative of 10 animals from two experiments and is gated on CD8 + lymphocytes. ( D ) pAls3-Tet staining of the draining LNs from an animal immunized with 20 μg recombinant Als3 in CFA 7 d postimmunization. ( E ) pAls3-Tet also was used to stain splenocytes ( upper panels ) and kidney-isolated leukocytes ( lower panels ) from infected mice at 7 d postinfection. Plots are representative of at least three animals from two independent experiments and are gated on B220 − CD11b − CD11c − CD3 + CD4 + CD8 − T cells. Data are pooled from two to four independent experiments.

    Journal: The Journal of Immunology Author Choice

    Article Title: Cutting Edge: Failure of Antigen-Specific CD4+ T Cell Recruitment to the Kidney during Systemic Candidiasis

    doi: 10.4049/jimmunol.1401675

    Figure Lengend Snippet: Ag-specific CD4 + T cells are not recruited to the infected kidney. Leukocytes from naive and infected kidneys ( A ) or small intestine ( B ) of mice, treated as in Fig. 1 , were analyzed by flow cytometry for the presence of OT.II (Vα2 + CD45.1 + ) cells. Example plots are gated on CD4 + lymphocytes and are representative of ≥12 animals from four experiments. Graph in (A) shows the frequency of OT.II cells in the kidney in naive ( n = 4) and infected ( n = 15) mice at day 3 postinfection. ( C ) Frequency of OT.I (Vα2 + CD45.1 + ) cells in naive ( n = 2) and infected ( n = 10) kidneys at day 3 postinfection. Example plot is representative of 10 animals from two experiments and is gated on CD8 + lymphocytes. ( D ) pAls3-Tet staining of the draining LNs from an animal immunized with 20 μg recombinant Als3 in CFA 7 d postimmunization. ( E ) pAls3-Tet also was used to stain splenocytes ( upper panels ) and kidney-isolated leukocytes ( lower panels ) from infected mice at 7 d postinfection. Plots are representative of at least three animals from two independent experiments and are gated on B220 − CD11b − CD11c − CD3 + CD4 + CD8 − T cells. Data are pooled from two to four independent experiments.

    Article Snippet: Multiplex PCR RNA was isolated from OT.II cells positively purified from the renal LNs using anti-CD45.1–biotin Ab and anti-biotin MicroBeads (Miltenyi Biotec), converted to cDNA using the SuperScript III synthesis system (Invitrogen), and used as a template in multiplex PCR reactions (Maxim Biotech), as per the manufacturers’ instructions.

    Techniques: Infection, Mouse Assay, Flow Cytometry, Cytometry, Staining, Recombinant, Isolation

    p53 modulates transcription of MnSOD as well as other target genes HepG2 cells were transfected with control siRNA or p53 siRNA along with MnSOD promoter (−555 to+24)-driven luciferase reporter vectors. After 24 h of co-transfection, cells were split into separate dishes and grown for another 24 h. A , cells were collected for luciferase activity as a measure of MnSOD gene transcription. B .” C , HepG2 cells were transfected with p53 expression vector (pcDNA3.1/p53, 4µg) or empty-vector (pcDNA3.1, equivalent amount), and total RNA was purified. The mRNA level of Bax gene under p53 overexpression conditions was determined by RT-PCR. A transcriptional off-target, the β-actin mRNA, was examined as an internal control. Statistical analyses were performed in three independent experiments **, p

    Journal: The Journal of biological chemistry

    Article Title: Specificity Protein 1-dependent p53-mediated Suppression of Human Manganese Superoxide Dismutase Gene Expression *

    doi: 10.1074/jbc.M601083200

    Figure Lengend Snippet: p53 modulates transcription of MnSOD as well as other target genes HepG2 cells were transfected with control siRNA or p53 siRNA along with MnSOD promoter (−555 to+24)-driven luciferase reporter vectors. After 24 h of co-transfection, cells were split into separate dishes and grown for another 24 h. A , cells were collected for luciferase activity as a measure of MnSOD gene transcription. B .” C , HepG2 cells were transfected with p53 expression vector (pcDNA3.1/p53, 4µg) or empty-vector (pcDNA3.1, equivalent amount), and total RNA was purified. The mRNA level of Bax gene under p53 overexpression conditions was determined by RT-PCR. A transcriptional off-target, the β-actin mRNA, was examined as an internal control. Statistical analyses were performed in three independent experiments **, p

    Article Snippet: For reverse transcription cDNA was generated using 2 µg of total RNA, oligo(dT) primer, and SuperScript™III first strand reverse transcriptase according to the manufacturer’s instructions (SuperScript™III first strand synthesis for RT-PCR, Invitrogen) in a total volume of 20µl.

    Techniques: Transfection, Luciferase, Cotransfection, Activity Assay, Expressing, Plasmid Preparation, Purification, Over Expression, Reverse Transcription Polymerase Chain Reaction

    Induced S-phase arrest of SNB-19 cells enhances ZIKV, but not DENV or WNV replication. (A) Schematic of two treatment conditions used to synchronize SNB-19 cells in S phase. Cells were initially treated with 2 mM thymidine for 16 h and then were released back into the cell cycle for 12 h. Following release, the cells were treated with either 2 mM thymidine or 12 μM aphidicolin for an additional 16 h to induce S-phase arrest prior to infection. (B) Representative flow cytometry analysis of SNB-19 cells treated as indicated in panel A. Samples were stained with propidium iodide (PI) and analyzed by flow cytometry. (C, D, and F) SNB-19 cells were infected for (C) 24 h, (D) 4 to 32 h, or (F) 12 to 24 h with ZIKV PR (MOI of 0.5), ZIKV M (MOI of 0.5), or DENV (MOI of 0.5). (C) Western blot images and quantification of intracellular viral protein expression. Viral protein was detected by anti-ZIKV NS1 or anti-DENV NS3 for each respective sample. Viral protein band intensities were normalized to the DMSO control, representing the mean from three biological replicates ± SD. (D and F) Time course of relative intracellular viral RNA copies in (D) ZIKV M - or (F) DENV-infected SNB-19 cells as measured by qPCR. Error bars are the mean ± SD from three biological replicates. (E and G) Infectivity titers of culture supernatants at (E) 48 h postinfection as measured by focus-forming units (ZIKV PR and ZIKV M [MOI of 0.5]) and at 36 h postinfection as measured by PFU (DENV [MOI of 0.5]) or (G) at 12 and 24 h postinfection as measured by PFU (WNV [MOI of 0.5]) on (E) Vero cells or (F) BHK cells. Error bars are the mean ± SD from three biological replicates. In panels C to G, * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, and **** indicates P ≤ 0.0001. (C, E, and G) One-way ANOVA. (D and F) Two-way ANOVA.

    Journal: Journal of Virology

    Article Title: Zika Virus Infection Induces DNA Damage Response in Human Neural Progenitors That Enhances Viral Replication

    doi: 10.1128/JVI.00638-19

    Figure Lengend Snippet: Induced S-phase arrest of SNB-19 cells enhances ZIKV, but not DENV or WNV replication. (A) Schematic of two treatment conditions used to synchronize SNB-19 cells in S phase. Cells were initially treated with 2 mM thymidine for 16 h and then were released back into the cell cycle for 12 h. Following release, the cells were treated with either 2 mM thymidine or 12 μM aphidicolin for an additional 16 h to induce S-phase arrest prior to infection. (B) Representative flow cytometry analysis of SNB-19 cells treated as indicated in panel A. Samples were stained with propidium iodide (PI) and analyzed by flow cytometry. (C, D, and F) SNB-19 cells were infected for (C) 24 h, (D) 4 to 32 h, or (F) 12 to 24 h with ZIKV PR (MOI of 0.5), ZIKV M (MOI of 0.5), or DENV (MOI of 0.5). (C) Western blot images and quantification of intracellular viral protein expression. Viral protein was detected by anti-ZIKV NS1 or anti-DENV NS3 for each respective sample. Viral protein band intensities were normalized to the DMSO control, representing the mean from three biological replicates ± SD. (D and F) Time course of relative intracellular viral RNA copies in (D) ZIKV M - or (F) DENV-infected SNB-19 cells as measured by qPCR. Error bars are the mean ± SD from three biological replicates. (E and G) Infectivity titers of culture supernatants at (E) 48 h postinfection as measured by focus-forming units (ZIKV PR and ZIKV M [MOI of 0.5]) and at 36 h postinfection as measured by PFU (DENV [MOI of 0.5]) or (G) at 12 and 24 h postinfection as measured by PFU (WNV [MOI of 0.5]) on (E) Vero cells or (F) BHK cells. Error bars are the mean ± SD from three biological replicates. In panels C to G, * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, and **** indicates P ≤ 0.0001. (C, E, and G) One-way ANOVA. (D and F) Two-way ANOVA.

    Article Snippet: Total cellular RNA was purified from mock-, ZIKVM -, or DENV-infected SNB-19 cells using an RNeasy Plus kit (Qiagen) per the manufacturer’s instructions. cDNA was synthesized from purified RNA using random hexamers and a SuperScript III First-Strand kit (Invitrogen).

    Techniques: Infection, Flow Cytometry, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction

    TLR2 −/− DCs show enhanced activation after L. braziliensis infection. WT and TLR2 −/− BMDCs were infected with L. braziliensis (Lb) and L. amazonensis (La) promastigotes at an 8:1 parasite-to-cell ratio for 24 h. Expression of DC surface maturation markers CD40 (A) and CD80 (B) was measured by FACS analysis. The levels of IL-12 p40 (C) and IL-10 (D) in the culture supernatants were assayed by ELISA. (E and F) DCs were infected at a 10:1 parasite-to-cell ratio for 4 h before RNA extraction. Total RNA was extracted and used for real-time RT-PCR measurement of SOCS1 (E) and SOCS3 (F) levels. Data are expressed as a fold change of expression relative to uninfected samples and normalized to the 18s ribosomal control. P 3 CSK 4 (Pam 3 CSK 4 ) was used as a quality control for TLR2. Data were pooled from three independent repeats and are shown in the plots. Statistically significant differences between the groups determined by one-way ANOVA and Student's t test are as follows: *, P

    Journal: Infection and Immunity

    Article Title: Distinct Roles for MyD88 and Toll-Like Receptor 2 during Leishmania braziliensis Infection in Mice ▿

    doi: 10.1128/IAI.00154-09

    Figure Lengend Snippet: TLR2 −/− DCs show enhanced activation after L. braziliensis infection. WT and TLR2 −/− BMDCs were infected with L. braziliensis (Lb) and L. amazonensis (La) promastigotes at an 8:1 parasite-to-cell ratio for 24 h. Expression of DC surface maturation markers CD40 (A) and CD80 (B) was measured by FACS analysis. The levels of IL-12 p40 (C) and IL-10 (D) in the culture supernatants were assayed by ELISA. (E and F) DCs were infected at a 10:1 parasite-to-cell ratio for 4 h before RNA extraction. Total RNA was extracted and used for real-time RT-PCR measurement of SOCS1 (E) and SOCS3 (F) levels. Data are expressed as a fold change of expression relative to uninfected samples and normalized to the 18s ribosomal control. P 3 CSK 4 (Pam 3 CSK 4 ) was used as a quality control for TLR2. Data were pooled from three independent repeats and are shown in the plots. Statistically significant differences between the groups determined by one-way ANOVA and Student's t test are as follows: *, P

    Article Snippet: For detecting suppressor or cytokine signaling 1 (SOCS1) and SOCS3 transcripts, cDNA was synthesized from 2 μg of total RNA by using the SuperScript III first-strand system (Invitrogen) primed with random hexamers.

    Techniques: Activation Assay, Infection, Expressing, FACS, Enzyme-linked Immunosorbent Assay, RNA Extraction, Quantitative RT-PCR

    STIM1 depletion reduces ER Ca 2+ content in T cells. ( A ) Expression of STIM1 ( Left ) and Orai1 ( Right ) in control and JP4-depleted Jurkat cells detected by immunoblotting and quantitative RT-PCR, respectively. The transcript data show mean ± SEM of triplicates. ( B ) STIM1 expression in control (Scr) or STIM1-depleted (STIM1 KD) Jurkat cells determined by immunoblotting. β-Actin was used as a loading control. ( C ) SOCE and ER Ca 2+ content measurements in control (Scr) or STIM1-depleted (STIM1 KD) Jurkat cells after passive store depletion with thapsigargin (TG) (1 μM) in Ca 2+ -free solution, and addition of 2 mM Ca 2+ -containing solution ( Left ). Traces show averaged (±SEM) responses from 30 to 50 Jurkat T cells. Bar graphs show change in ER Ca 2+ content and SOCE (±SEM) from three independent experiments. Control (Scr) and STIM1-depleted (KD) Jurkat cells were treated with ionomycin (1 μM, iono) in Ca 2+ -free solution to measure the ER Ca 2+ content ( Right two panels). Traces show averaged (±SEM) responses from 30 to 50 cells, and bar graph shows change in ER Ca 2+ content (±SEM) from three independent experiments. * P

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

    Article Title: Junctophilin-4, a component of the endoplasmic reticulum–plasma membrane junctions, regulates Ca2+ dynamics in T cells

    doi: 10.1073/pnas.1524229113

    Figure Lengend Snippet: STIM1 depletion reduces ER Ca 2+ content in T cells. ( A ) Expression of STIM1 ( Left ) and Orai1 ( Right ) in control and JP4-depleted Jurkat cells detected by immunoblotting and quantitative RT-PCR, respectively. The transcript data show mean ± SEM of triplicates. ( B ) STIM1 expression in control (Scr) or STIM1-depleted (STIM1 KD) Jurkat cells determined by immunoblotting. β-Actin was used as a loading control. ( C ) SOCE and ER Ca 2+ content measurements in control (Scr) or STIM1-depleted (STIM1 KD) Jurkat cells after passive store depletion with thapsigargin (TG) (1 μM) in Ca 2+ -free solution, and addition of 2 mM Ca 2+ -containing solution ( Left ). Traces show averaged (±SEM) responses from 30 to 50 Jurkat T cells. Bar graphs show change in ER Ca 2+ content and SOCE (±SEM) from three independent experiments. Control (Scr) and STIM1-depleted (KD) Jurkat cells were treated with ionomycin (1 μM, iono) in Ca 2+ -free solution to measure the ER Ca 2+ content ( Right two panels). Traces show averaged (±SEM) responses from 30 to 50 cells, and bar graph shows change in ER Ca 2+ content (±SEM) from three independent experiments. * P

    Article Snippet: For RT-PCR, purified RNA was oligo(dT)-primed for first-strand cDNA synthesis (Superscript III kit; Invitrogen).

    Techniques: Expressing, Quantitative RT-PCR