site directed mutagenesis  (Agilent technologies)

 
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    4500 Series Portable FTIR
    Description:
    The Agilent 4500 Portable FTIR Spectrometer is a rugged non lab analyzer that support efforts associated with on the spot analysis of incoming materials and outgoing finished products in the chemical food and polymer industries It is also ideal for proactive maintenance programs of high value equipment and machinery in construction and power production industries Exceedingly compact easy to use and rugged this portable FTIR spectrometer is a perfect match for applications that require high quality answers quickly The combination of optics designed for reliability in non lab environments innovative sampling interfaces and fit for purpose software provides answers for liquid and solid samples at the sample site
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    4500-SERIES-PORTABLE-FTIR
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    Products Ftir Ftir Compact Portable Systems 4500 Series Portable Ftir
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    Agilent technologies site directed mutagenesis
    The Agilent 4500 Portable FTIR Spectrometer is a rugged non lab analyzer that support efforts associated with on the spot analysis of incoming materials and outgoing finished products in the chemical food and polymer industries It is also ideal for proactive maintenance programs of high value equipment and machinery in construction and power production industries Exceedingly compact easy to use and rugged this portable FTIR spectrometer is a perfect match for applications that require high quality answers quickly The combination of optics designed for reliability in non lab environments innovative sampling interfaces and fit for purpose software provides answers for liquid and solid samples at the sample site
    https://www.bioz.com/result/site directed mutagenesis/product/Agilent technologies
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    site directed mutagenesis - by Bioz Stars, 2021-05
    86/100 stars

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    Article Title: Measuring Body Composition in Low-Resource Settings across the Life Course
    Article Snippet: Rein A, Higgins F, Preston T. [06-05-2015]; Application of the Agilent 4500 Series FTIR to the Stable Isotope Technique for Assessing Intake of Human Milk in Breastfed Infants.

    Article Title: Rapid Analysis of Milk Using Low-Cost Pocket-Size NIR Spectrometers and Multivariate Analysis
    Article Snippet: Various commercial hand-held devices, such as the Polychromix PHAZIR™ (Polychromix Inc., Wilmington, MA, USA) [ , ], 4200 FlexScan FTIR (Agilent Technologies Inc., Danbury, CT, USA) [ ], Micro-NIR 1700 (JDSU, Milpitas, CA, USA) [ , ] or the Agilent 4500 portable ATR-FTIR (Agilent Technologies, Inc., Santa Clara, CA, USA) [ ] have been used for milk content analysis.

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    Article Title: Infrared spectroscopy coupled to cloud-based data management as a tool to diagnose malaria: a pilot study in a malaria-endemic country
    Article Snippet: Malaria diagnosis using ATR-FTIR spectroscopy .. ATR-FTIR spectroscopy data acquisition The methodology for data acquisition using ATR-FTIR spectroscopy from methanol fixed packed red blood cells was identical to that previously described [ ], except that a portable Agilent 4500 ATR-FTIR spectrometer was employed. ..

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    Agilent technologies pcr based site directed mutagenesis kit
    Methylation status of the <t>miR-520c</t> regulatory region and expression of miR-520c-3p and its target S100A4 in a cohort of CRC tumor specimens ( A ) Methylation specific <t>PCR</t> products of metachronous metastasis positive and metachronous negative CRC tumor specimens were analyzed using agarose gel electrophoresis. The miR-520c regulatory element was methylated in all specimens. The two cell lines SW620 and Colo206f served as internal controls. ( B ) miR-520c-3p expression in metachronous metastasis positive ( n = 10) and negative ( n = 10) CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. ( C , D ) miR-520c-3p and S100A4 expression in CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. RPII and RNUB6 served as internal controls.
    Pcr Based Site Directed Mutagenesis Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based site directed mutagenesis kit/product/Agilent technologies
    Average 99 stars, based on 1 article reviews
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    86
    Agilent technologies pcr based site directed mutagenesis
    Effect of Aβ on ADAM10 and miR-140-5p (A) Neuronal SHSY5Y cells were transfected with ADAM10 promoter (ADAM10p) and ADAM10 <t>promoter-3’UTR</t> (ADAM10p+3’utr) reporter constructs, 48 h post-transfection is followed by treatment with Aβ (5 µM) for 24 hrs. Results are presented as Firefly/Renilla luciferase activity of the respective constructs with or without Aβ as control. Data represent at least six independent experiments with triplicate measurements. (B) Neuronal SHSY5Y cells with miR-140-5p expression either without or with Aβ (5 µM) treatment and transfected with miRNA-140-5p inhibitor. MiRNA-140-5p expression was measured by <t>qRT-PCR</t> and represented as fold change compared with untreated control. Results are represented by 2^-ΔΔCT. Black line indicates Fisher exact t-test p-value
    Pcr Based Site Directed Mutagenesis, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based site directed mutagenesis/product/Agilent technologies
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    86
    Agilent technologies srsf2 mutants
    The <t>SRSF2</t> P95H mutation alters SRSF2 in vivo RNA motif specificity (a) Top enriched motifs for WT and P95H SRSF2 binding sites, identified by discriminative analysis of kmer composition. Corresponding p-values are displayed on top of each logo. (b) Relative enrichment of the SSNG (S=C/G, N=C/G/A/U) RNA consensus motifs in RNA regions preferentially bound by WT versus P95H SRSF2. The number of motif occurrences and differential enrichment p-values are displayed for each bar.
    Srsf2 Mutants, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Agilent technologies quikchange xl multi site directed mutagenesis kit
    Summary of PAR2 C-tail residue mutations ( A) Cartoon depicting PAR2 with palmitoylation site (green) and phosphorylation sites (blue) shown (generated with GPCRdb) ( 67 , 68 ). (B) Mutant receptor clones generated using Agilent <t>QuikChange</t> XL site-directed mutagenesis. Sequences of the wildtype PAR2 and mutant PAR2 receptor C-tail constructs (mutations are shown underlined). PAR2C 361 A mutation was generated to study the effects of this residue on signalling endpoints. PAR2S 383-386 A and PAR2S 387- T 392A mutants were generated to determine the effects of partial mutation of the serine/threonine rich region of the PAR2 C-tail. A combined mutant of PAR2S 383-385 A, S 387- T 392 A was also generated to determine the effect on mutation of serine/threonine rich C-tail motif on signalling.
    Quikchange Xl Multi Site Directed Mutagenesis Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quikchange xl multi site directed mutagenesis kit/product/Agilent technologies
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    Methylation status of the miR-520c regulatory region and expression of miR-520c-3p and its target S100A4 in a cohort of CRC tumor specimens ( A ) Methylation specific PCR products of metachronous metastasis positive and metachronous negative CRC tumor specimens were analyzed using agarose gel electrophoresis. The miR-520c regulatory element was methylated in all specimens. The two cell lines SW620 and Colo206f served as internal controls. ( B ) miR-520c-3p expression in metachronous metastasis positive ( n = 10) and negative ( n = 10) CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. ( C , D ) miR-520c-3p and S100A4 expression in CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. RPII and RNUB6 served as internal controls.

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: Methylation status of the miR-520c regulatory region and expression of miR-520c-3p and its target S100A4 in a cohort of CRC tumor specimens ( A ) Methylation specific PCR products of metachronous metastasis positive and metachronous negative CRC tumor specimens were analyzed using agarose gel electrophoresis. The miR-520c regulatory element was methylated in all specimens. The two cell lines SW620 and Colo206f served as internal controls. ( B ) miR-520c-3p expression in metachronous metastasis positive ( n = 10) and negative ( n = 10) CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. ( C , D ) miR-520c-3p and S100A4 expression in CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. RPII and RNUB6 served as internal controls.

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Methylation, Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR

    miR-505-5p and miR-520c-3p inhibit the S100A4 mediated migration and invasion in vitro ( A ) HCT116 cells were co-transfected with control-miR, miR-505-5p and miR-520c-3p along with either vector-control or -S100A4, respectively. After 48 h cells were plated on top of the Boyden chambers for migration and matrigel-coated Boyden chambers for invasion. After 16 h the migrated or invaded cells were measured as described in the materials and methods. ( B ) SW620 cells were co-transfected with control-miR, miR-505-5p and miR-520c-3p along with either si-RNA-control (si-control) or si-RNA-S100A4 (si-S100A4), respectively. After 48 h the cells were used for migration and invasion assay as stated above. ( C , D ) Transfection efficiency of ectopic overexpression and knock-down of S100A4 on mRNA level was analyzed using qRT-PCR. ( E , F ) S100A4 protein expression after transfection was analyzed by Western blots. RPII was used as internal control for S100A4 mRNA expression and β-actin for Western blotting. ( *p

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: miR-505-5p and miR-520c-3p inhibit the S100A4 mediated migration and invasion in vitro ( A ) HCT116 cells were co-transfected with control-miR, miR-505-5p and miR-520c-3p along with either vector-control or -S100A4, respectively. After 48 h cells were plated on top of the Boyden chambers for migration and matrigel-coated Boyden chambers for invasion. After 16 h the migrated or invaded cells were measured as described in the materials and methods. ( B ) SW620 cells were co-transfected with control-miR, miR-505-5p and miR-520c-3p along with either si-RNA-control (si-control) or si-RNA-S100A4 (si-S100A4), respectively. After 48 h the cells were used for migration and invasion assay as stated above. ( C , D ) Transfection efficiency of ectopic overexpression and knock-down of S100A4 on mRNA level was analyzed using qRT-PCR. ( E , F ) S100A4 protein expression after transfection was analyzed by Western blots. RPII was used as internal control for S100A4 mRNA expression and β-actin for Western blotting. ( *p

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Migration, In Vitro, Transfection, Plasmid Preparation, Invasion Assay, Over Expression, Quantitative RT-PCR, Expressing, Western Blot

    miR-520c regulatory region is hypermethylated in CRC cell lines and treatment with 5-Aza induces the expression of miR-520c-3p and downregulates S100A4 expression ( A , B ) SW480, SW620 and HCT116 cells were treated with 5-Aza (2 mM) for 3 days. qRT-PCR and Western blots were performed to analyze the impact of 5-Aza treatment on miR-520c-3p and S100A4 expression. RNUB6, RPII and β-actin served as internal controls. ( C ) HCT116 and SW620 cells transfected with the wild type S100A4-3′-UTR were treated with 5-Aza (2 μM) for 24 h and the luciferase activity was measured. Renilla luciferase activity was used for normalization. Percentage luciferase activity was significantly reduced after 5-Aza. ( *p

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: miR-520c regulatory region is hypermethylated in CRC cell lines and treatment with 5-Aza induces the expression of miR-520c-3p and downregulates S100A4 expression ( A , B ) SW480, SW620 and HCT116 cells were treated with 5-Aza (2 mM) for 3 days. qRT-PCR and Western blots were performed to analyze the impact of 5-Aza treatment on miR-520c-3p and S100A4 expression. RNUB6, RPII and β-actin served as internal controls. ( C ) HCT116 and SW620 cells transfected with the wild type S100A4-3′-UTR were treated with 5-Aza (2 μM) for 24 h and the luciferase activity was measured. Renilla luciferase activity was used for normalization. Percentage luciferase activity was significantly reduced after 5-Aza. ( *p

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Activity Assay

    COBRA and bisulfite sequencing analysis of miR-520c regulatory region ( A , B ) COBRA analysis using the restriction enzyme BstUI was performed to estimate the methylation status of miR-520c-3p in CRC cell lines. Colo206f, Colo320DM and WiDr were also analyzed after 5-Aza (2 μm) treatment for 3 days. ( C ) Colo320DM cells were treated with 2 μM 5-Aza for 3 days and the methylation of miR-520c-3p was analyzed by bisulfite sequencing. From each group 10 representative clones were sequenced. Each circle represents CpG islands in the sequenced region, and the black circle represents the methylated and the empty white circle represents unmethylated CpG island. ( D , E ) qRT-PCR of miR-520c-3p, S100A4 and Western blot analysis of S100A4 were performed after 5-Aza treatment of Colo206f, Colo320DM for 3 days (20 μg of total protein used for Western blot) and WiDr (5 μg of total protein used for Western blot) cell lines (from low to moderate S100A4 expressing cells). RNUB6, RPII and β-actin served as internal controls. ( *p

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: COBRA and bisulfite sequencing analysis of miR-520c regulatory region ( A , B ) COBRA analysis using the restriction enzyme BstUI was performed to estimate the methylation status of miR-520c-3p in CRC cell lines. Colo206f, Colo320DM and WiDr were also analyzed after 5-Aza (2 μm) treatment for 3 days. ( C ) Colo320DM cells were treated with 2 μM 5-Aza for 3 days and the methylation of miR-520c-3p was analyzed by bisulfite sequencing. From each group 10 representative clones were sequenced. Each circle represents CpG islands in the sequenced region, and the black circle represents the methylated and the empty white circle represents unmethylated CpG island. ( D , E ) qRT-PCR of miR-520c-3p, S100A4 and Western blot analysis of S100A4 were performed after 5-Aza treatment of Colo206f, Colo320DM for 3 days (20 μg of total protein used for Western blot) and WiDr (5 μg of total protein used for Western blot) cell lines (from low to moderate S100A4 expressing cells). RNUB6, RPII and β-actin served as internal controls. ( *p

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Combined Bisulfite Restriction Analysis Assay, Methylation Sequencing, Methylation, Clone Assay, Quantitative RT-PCR, Western Blot, Expressing

    Overexpression of miR-520c-3p inhibits metastasis formation in vivo ( A ) HCT116 cells stably expressing miR-520c-3p or control-miR were generated. The overexpression of the miR was quantified by qRT-PCR and compared to control-miR. The expression of the target gene S100A4 in the miR overexpressing cells was downregulated as indicated by Western blotting. ( B ) The cells were intrasplenically injected in SCID beige mice and sacrificed after 20 days. The expression of miR-520c-3p in the shock-frozen primary tumor in the spleen was quantified by qRT-PCR. ( C ) Metastasized cells into the liver of the animals were analyzed using specific primers to exclusively detect HMD by qRT-PCR. Less amounts of HMD were detectable in the liver sections of the miR-520c-3p overexpressing group. ( D ) The cells were intrasplenically injected in SCID beige mice and tumor and metastasis formation was monitored by bioluminescence imaging. Representative mice from each group are shown on the day the animals were sacrificed. ( *p

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: Overexpression of miR-520c-3p inhibits metastasis formation in vivo ( A ) HCT116 cells stably expressing miR-520c-3p or control-miR were generated. The overexpression of the miR was quantified by qRT-PCR and compared to control-miR. The expression of the target gene S100A4 in the miR overexpressing cells was downregulated as indicated by Western blotting. ( B ) The cells were intrasplenically injected in SCID beige mice and sacrificed after 20 days. The expression of miR-520c-3p in the shock-frozen primary tumor in the spleen was quantified by qRT-PCR. ( C ) Metastasized cells into the liver of the animals were analyzed using specific primers to exclusively detect HMD by qRT-PCR. Less amounts of HMD were detectable in the liver sections of the miR-520c-3p overexpressing group. ( D ) The cells were intrasplenically injected in SCID beige mice and tumor and metastasis formation was monitored by bioluminescence imaging. Representative mice from each group are shown on the day the animals were sacrificed. ( *p

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Generated, Quantitative RT-PCR, Western Blot, Injection, Mouse Assay, Imaging

    miR-505-5p and miR-520c-3p target the S100A4-3′UTR, and downregulate S100A4 expression ( A ) The seed sequences for miR-505-5p and miR-520c-3p in S100A4-3′-UTR (wild type) were detected by in silico predictions. The wild type and mutated seed sequence were cloned into the dual-luciferase vector and luciferase assays were performed to show direct regulation of the S100A4-3′-UTR by these miRs. ( B ) Luciferase reporter assays of HCT116 and SW620 cells transfected with control-miR (Control), miR-505-5p, anti-miR-505-5p, miR-520c-3p, anti-miR-520c-3p and co-transfected with either S100A4-3′-UTR wild type or the mutated constructs were performed. Renilla luciferase values of the vector were used for normalization. Percent luciferase activity was calculated based on the control values. ( C ) S100A4 mRNA expression was quantified using qRT-PCR and RPII was used as internal control. ( D ) S100A4 Western blotting was performed and bands were densitometrically quantified by normalizing to the internal control β-actin. Mean values of triplicates were represented. ( *p

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: miR-505-5p and miR-520c-3p target the S100A4-3′UTR, and downregulate S100A4 expression ( A ) The seed sequences for miR-505-5p and miR-520c-3p in S100A4-3′-UTR (wild type) were detected by in silico predictions. The wild type and mutated seed sequence were cloned into the dual-luciferase vector and luciferase assays were performed to show direct regulation of the S100A4-3′-UTR by these miRs. ( B ) Luciferase reporter assays of HCT116 and SW620 cells transfected with control-miR (Control), miR-505-5p, anti-miR-505-5p, miR-520c-3p, anti-miR-520c-3p and co-transfected with either S100A4-3′-UTR wild type or the mutated constructs were performed. Renilla luciferase values of the vector were used for normalization. Percent luciferase activity was calculated based on the control values. ( C ) S100A4 mRNA expression was quantified using qRT-PCR and RPII was used as internal control. ( D ) S100A4 Western blotting was performed and bands were densitometrically quantified by normalizing to the internal control β-actin. Mean values of triplicates were represented. ( *p

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Expressing, In Silico, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Construct, Activity Assay, Quantitative RT-PCR, Western Blot

    Effect of Aβ on ADAM10 and miR-140-5p (A) Neuronal SHSY5Y cells were transfected with ADAM10 promoter (ADAM10p) and ADAM10 promoter-3’UTR (ADAM10p+3’utr) reporter constructs, 48 h post-transfection is followed by treatment with Aβ (5 µM) for 24 hrs. Results are presented as Firefly/Renilla luciferase activity of the respective constructs with or without Aβ as control. Data represent at least six independent experiments with triplicate measurements. (B) Neuronal SHSY5Y cells with miR-140-5p expression either without or with Aβ (5 µM) treatment and transfected with miRNA-140-5p inhibitor. MiRNA-140-5p expression was measured by qRT-PCR and represented as fold change compared with untreated control. Results are represented by 2^-ΔΔCT. Black line indicates Fisher exact t-test p-value

    Journal: Neurobiology of aging

    Article Title: Regulation of ADAM10 by miR-140-5p and potential relevance for Alzheimer’s Disease

    doi: 10.1016/j.neurobiolaging.2017.11.007

    Figure Lengend Snippet: Effect of Aβ on ADAM10 and miR-140-5p (A) Neuronal SHSY5Y cells were transfected with ADAM10 promoter (ADAM10p) and ADAM10 promoter-3’UTR (ADAM10p+3’utr) reporter constructs, 48 h post-transfection is followed by treatment with Aβ (5 µM) for 24 hrs. Results are presented as Firefly/Renilla luciferase activity of the respective constructs with or without Aβ as control. Data represent at least six independent experiments with triplicate measurements. (B) Neuronal SHSY5Y cells with miR-140-5p expression either without or with Aβ (5 µM) treatment and transfected with miRNA-140-5p inhibitor. MiRNA-140-5p expression was measured by qRT-PCR and represented as fold change compared with untreated control. Results are represented by 2^-ΔΔCT. Black line indicates Fisher exact t-test p-value

    Article Snippet: ADAM10 and SOX2 reporter constructs with mutations in promoter or 3’UTR were generated by incorporating mutations into the miR-140 binding site on ADAM10 3’UTR and SOX2 3’UTR as well as in SOX2 binding site on ADAM10 promoter by PCR-based site directed mutagenesis using Pfu Turbo DNA polymerase (Quickchange II Site Directed Mutagenesis Kit; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol and were verified by sequencing.

    Techniques: Transfection, Construct, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR

    MiRNA expression levels in human AD hippocampus compared to controls (A) The expression profile of ADAM10 candidate miRNAs in microarray represented by 2^-ΔΔCT`. (B) Quantitative RT-PCR analysis of ADAM10 candidate miRNA-140, miRNA-182, miRNA-194 and control miRNA-126 in Control vs AD. Data represent three independent experiments with triplicate measurements. All p-values were corrected with the Bonferroni correction for multiple comparisons.

    Journal: Neurobiology of aging

    Article Title: Regulation of ADAM10 by miR-140-5p and potential relevance for Alzheimer’s Disease

    doi: 10.1016/j.neurobiolaging.2017.11.007

    Figure Lengend Snippet: MiRNA expression levels in human AD hippocampus compared to controls (A) The expression profile of ADAM10 candidate miRNAs in microarray represented by 2^-ΔΔCT`. (B) Quantitative RT-PCR analysis of ADAM10 candidate miRNA-140, miRNA-182, miRNA-194 and control miRNA-126 in Control vs AD. Data represent three independent experiments with triplicate measurements. All p-values were corrected with the Bonferroni correction for multiple comparisons.

    Article Snippet: ADAM10 and SOX2 reporter constructs with mutations in promoter or 3’UTR were generated by incorporating mutations into the miR-140 binding site on ADAM10 3’UTR and SOX2 3’UTR as well as in SOX2 binding site on ADAM10 promoter by PCR-based site directed mutagenesis using Pfu Turbo DNA polymerase (Quickchange II Site Directed Mutagenesis Kit; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol and were verified by sequencing.

    Techniques: Expressing, Microarray, Quantitative RT-PCR

    The SRSF2 P95H mutation alters SRSF2 in vivo RNA motif specificity (a) Top enriched motifs for WT and P95H SRSF2 binding sites, identified by discriminative analysis of kmer composition. Corresponding p-values are displayed on top of each logo. (b) Relative enrichment of the SSNG (S=C/G, N=C/G/A/U) RNA consensus motifs in RNA regions preferentially bound by WT versus P95H SRSF2. The number of motif occurrences and differential enrichment p-values are displayed for each bar.

    Journal: Leukemia

    Article Title: SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells

    doi: 10.1038/s41375-018-0152-7

    Figure Lengend Snippet: The SRSF2 P95H mutation alters SRSF2 in vivo RNA motif specificity (a) Top enriched motifs for WT and P95H SRSF2 binding sites, identified by discriminative analysis of kmer composition. Corresponding p-values are displayed on top of each logo. (b) Relative enrichment of the SSNG (S=C/G, N=C/G/A/U) RNA consensus motifs in RNA regions preferentially bound by WT versus P95H SRSF2. The number of motif occurrences and differential enrichment p-values are displayed for each bar.

    Article Snippet: Site-directed mutagenesis was performed to obtain SRSF2 mutants per standard protocol (Agilent Technologies).

    Techniques: Mutagenesis, In Vivo, Binding Assay

    SRSF2 mutations results in differential binding and splicing of HNRNP proteins (a–c) Differential binding and splicing in HNRNP proteins is shown for HNRNPA2B1 ( a ), HNRNPH1 ( b ), and HNRNPM ( c ). Left panels: transcript maps showing WT (red) and P95H (cyan) SRSF2 binding profiles. The maps display mean normalized HITS-CLIP signal with nucleotide resolution. Standard errors for each position are shown as ribbons under mean lines. Crosslinking-induced deletions are marked in black. Exon boundaries are represented as vertical dotted lines. Differential interaction sites are highlighted on the transcript. Center panels: RT-PCR capturing differentially bound exons was performed in 3–4 replicates in HEL cells with or without doxycycline induction of SRSF2 WT or P95H expression, and with or without knockdown of endogenous SRSF2, and in human fetal liver CD34+ cells transduced with empty, SRSF2 WT or SRSF2 P95H expressing lentivirus. Right panels: primary patient derived samples - alternative splice events were quantified in normal CD34+ (n=2), WT MDS/AML (n=6), and MUT MDS/AML (n=6) samples. The magnitude of the alternative splice event in % was calculated as ratio of alternative splice event to total expression. Considered bands are marked by an asterisk (*). Predicted band sizes and transcript sizes are indicated to the right of the gel. (d) Direct differential splicing of the HNRNPA2B1 exon 9 by WT vs P95H SRSF2 verified by a minigene splicing assay. RG6-HNRNPA2B1 plasmid was co-transfected with empty vector, SRSF2 WT, SRSF2 P95H/L/R, and control or SRSF2 siRNA. Alternative splicing of exon 9 was determined via semi-quantitative PCR by measuring the ratio of alternative exon exclusion over total (exclusion+inclusion) band intensities (n=3). (e) Colony Forming Unit Assay for control cells (siNTC), cells silenced for HNRNPM or HNRNPH1, cells induced to splice out HNRNPA2B1 exon 9, and cells treated for all three modifications together (siALL). The same number of cells was plated in all experiments. The total number and the composition of colonies is displayed. Total colony numbers and colony type percentages were compared to the control (siNTC). Standard errors (SE) for colony numbers are displayed. In panels ( a–e) , significance values were determined by one way ANOVA with Sidák Post Hoc Test (*P

    Journal: Leukemia

    Article Title: SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells

    doi: 10.1038/s41375-018-0152-7

    Figure Lengend Snippet: SRSF2 mutations results in differential binding and splicing of HNRNP proteins (a–c) Differential binding and splicing in HNRNP proteins is shown for HNRNPA2B1 ( a ), HNRNPH1 ( b ), and HNRNPM ( c ). Left panels: transcript maps showing WT (red) and P95H (cyan) SRSF2 binding profiles. The maps display mean normalized HITS-CLIP signal with nucleotide resolution. Standard errors for each position are shown as ribbons under mean lines. Crosslinking-induced deletions are marked in black. Exon boundaries are represented as vertical dotted lines. Differential interaction sites are highlighted on the transcript. Center panels: RT-PCR capturing differentially bound exons was performed in 3–4 replicates in HEL cells with or without doxycycline induction of SRSF2 WT or P95H expression, and with or without knockdown of endogenous SRSF2, and in human fetal liver CD34+ cells transduced with empty, SRSF2 WT or SRSF2 P95H expressing lentivirus. Right panels: primary patient derived samples - alternative splice events were quantified in normal CD34+ (n=2), WT MDS/AML (n=6), and MUT MDS/AML (n=6) samples. The magnitude of the alternative splice event in % was calculated as ratio of alternative splice event to total expression. Considered bands are marked by an asterisk (*). Predicted band sizes and transcript sizes are indicated to the right of the gel. (d) Direct differential splicing of the HNRNPA2B1 exon 9 by WT vs P95H SRSF2 verified by a minigene splicing assay. RG6-HNRNPA2B1 plasmid was co-transfected with empty vector, SRSF2 WT, SRSF2 P95H/L/R, and control or SRSF2 siRNA. Alternative splicing of exon 9 was determined via semi-quantitative PCR by measuring the ratio of alternative exon exclusion over total (exclusion+inclusion) band intensities (n=3). (e) Colony Forming Unit Assay for control cells (siNTC), cells silenced for HNRNPM or HNRNPH1, cells induced to splice out HNRNPA2B1 exon 9, and cells treated for all three modifications together (siALL). The same number of cells was plated in all experiments. The total number and the composition of colonies is displayed. Total colony numbers and colony type percentages were compared to the control (siNTC). Standard errors (SE) for colony numbers are displayed. In panels ( a–e) , significance values were determined by one way ANOVA with Sidák Post Hoc Test (*P

    Article Snippet: Site-directed mutagenesis was performed to obtain SRSF2 mutants per standard protocol (Agilent Technologies).

    Techniques: Binding Assay, Cross-linking Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transduction, Derivative Assay, Splicing Assay, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Colony-forming Unit Assay

    The SRSF2 P95H mutation alters SRSF2 in vivo RNA interactome (a) Overview of the HITS-CLIP procedure. Top: generation of lentiviral vector constructs expressing C-terminally Flag-tagged SRSF2 WT and P95H in a doxycycline inducible manner. Center: HITS-CLIP key experimental steps. Bottom: computational identification of differentially bound regions, preferentially bound by WT (in red) or by P95H (in cyan) SRSF2. (b) Dose dependent inducible expression of Flag-tagged SRSF2 (WT and P95H). (c) Colony Forming Unit Assay for control CD34+ cells (mCherry), and cells with transient induction of SRSF2 WT or P95H. Left panel: total number of colonies. Right panel: composition of colonies (mean values + SEM). Total colony numbers and colony type percentages were compared between WT and P95H by two-tailed t-test (*P

    Journal: Leukemia

    Article Title: SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells

    doi: 10.1038/s41375-018-0152-7

    Figure Lengend Snippet: The SRSF2 P95H mutation alters SRSF2 in vivo RNA interactome (a) Overview of the HITS-CLIP procedure. Top: generation of lentiviral vector constructs expressing C-terminally Flag-tagged SRSF2 WT and P95H in a doxycycline inducible manner. Center: HITS-CLIP key experimental steps. Bottom: computational identification of differentially bound regions, preferentially bound by WT (in red) or by P95H (in cyan) SRSF2. (b) Dose dependent inducible expression of Flag-tagged SRSF2 (WT and P95H). (c) Colony Forming Unit Assay for control CD34+ cells (mCherry), and cells with transient induction of SRSF2 WT or P95H. Left panel: total number of colonies. Right panel: composition of colonies (mean values + SEM). Total colony numbers and colony type percentages were compared between WT and P95H by two-tailed t-test (*P

    Article Snippet: Site-directed mutagenesis was performed to obtain SRSF2 mutants per standard protocol (Agilent Technologies).

    Techniques: Mutagenesis, In Vivo, Cross-linking Immunoprecipitation, Plasmid Preparation, Construct, Expressing, Colony-forming Unit Assay, Two Tailed Test

    Differentially bound SRSF2 targets are enriched in RNA binding and splicing genes (a) Functional annotation enrichment analysis of differentially bound transcripts by WT and P95H SRSF2. The number of genes belonging to each category is displayed. (b) Protein-protein interaction network of SRSF2 RNA interactors associated with splicing. The size of each node is proportional to the number of differential SRSF2 binding sites: WT (in red), P95H (in cyan) or both (in violet).

    Journal: Leukemia

    Article Title: SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells

    doi: 10.1038/s41375-018-0152-7

    Figure Lengend Snippet: Differentially bound SRSF2 targets are enriched in RNA binding and splicing genes (a) Functional annotation enrichment analysis of differentially bound transcripts by WT and P95H SRSF2. The number of genes belonging to each category is displayed. (b) Protein-protein interaction network of SRSF2 RNA interactors associated with splicing. The size of each node is proportional to the number of differential SRSF2 binding sites: WT (in red), P95H (in cyan) or both (in violet).

    Article Snippet: Site-directed mutagenesis was performed to obtain SRSF2 mutants per standard protocol (Agilent Technologies).

    Techniques: RNA Binding Assay, Functional Assay, Binding Assay

    SRSF2 P95H mutations promote alternative splicing with inclusion of CCNG rich exons and enrichment in RNA binding and splicing genes (a) Determination of differential alternative splice events via rMATS analysis in HEL cells engineered to express SRSF2 WT vs P95H. Left panel: five classes of alternative splicing events were considered: cassette exon (CE), alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE) and retained intron (RI). Right panel: the number of significant events with more inclusion in WT or P95H cells is displayed. (b) Relative enrichment of the SSNG (S=C/G, N=C/G/A/T) RNA consensus motifs in cassette exons preferentially spliced in WT vs preferentially spliced in P95H SRSF2 expressing cells. The number of motif occurrences and enrichment p-values are displayed for each bar. (c) Overlap between genes with differential binding and differential splicing in HEL cells expressing either WT or P95H SRSF2. The significance of the overlap is displayed. (d) Functional annotation enrichment analysis of differentially spliced genes in WT vs P95H HEL cells. The number of genes falling into each category is indicated beside each bar.

    Journal: Leukemia

    Article Title: SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells

    doi: 10.1038/s41375-018-0152-7

    Figure Lengend Snippet: SRSF2 P95H mutations promote alternative splicing with inclusion of CCNG rich exons and enrichment in RNA binding and splicing genes (a) Determination of differential alternative splice events via rMATS analysis in HEL cells engineered to express SRSF2 WT vs P95H. Left panel: five classes of alternative splicing events were considered: cassette exon (CE), alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE) and retained intron (RI). Right panel: the number of significant events with more inclusion in WT or P95H cells is displayed. (b) Relative enrichment of the SSNG (S=C/G, N=C/G/A/T) RNA consensus motifs in cassette exons preferentially spliced in WT vs preferentially spliced in P95H SRSF2 expressing cells. The number of motif occurrences and enrichment p-values are displayed for each bar. (c) Overlap between genes with differential binding and differential splicing in HEL cells expressing either WT or P95H SRSF2. The significance of the overlap is displayed. (d) Functional annotation enrichment analysis of differentially spliced genes in WT vs P95H HEL cells. The number of genes falling into each category is indicated beside each bar.

    Article Snippet: Site-directed mutagenesis was performed to obtain SRSF2 mutants per standard protocol (Agilent Technologies).

    Techniques: RNA Binding Assay, Expressing, Binding Assay, Functional Assay

    Summary of PAR2 C-tail residue mutations ( A) Cartoon depicting PAR2 with palmitoylation site (green) and phosphorylation sites (blue) shown (generated with GPCRdb) ( 67 , 68 ). (B) Mutant receptor clones generated using Agilent QuikChange XL site-directed mutagenesis. Sequences of the wildtype PAR2 and mutant PAR2 receptor C-tail constructs (mutations are shown underlined). PAR2C 361 A mutation was generated to study the effects of this residue on signalling endpoints. PAR2S 383-386 A and PAR2S 387- T 392A mutants were generated to determine the effects of partial mutation of the serine/threonine rich region of the PAR2 C-tail. A combined mutant of PAR2S 383-385 A, S 387- T 392 A was also generated to determine the effect on mutation of serine/threonine rich C-tail motif on signalling.

    Journal: bioRxiv

    Article Title: Role of the C-terminal tail in regulating Proteinase Activated Receptor 2 (PAR2) signalling

    doi: 10.1101/2020.03.02.973842

    Figure Lengend Snippet: Summary of PAR2 C-tail residue mutations ( A) Cartoon depicting PAR2 with palmitoylation site (green) and phosphorylation sites (blue) shown (generated with GPCRdb) ( 67 , 68 ). (B) Mutant receptor clones generated using Agilent QuikChange XL site-directed mutagenesis. Sequences of the wildtype PAR2 and mutant PAR2 receptor C-tail constructs (mutations are shown underlined). PAR2C 361 A mutation was generated to study the effects of this residue on signalling endpoints. PAR2S 383-386 A and PAR2S 387- T 392A mutants were generated to determine the effects of partial mutation of the serine/threonine rich region of the PAR2 C-tail. A combined mutant of PAR2S 383-385 A, S 387- T 392 A was also generated to determine the effect on mutation of serine/threonine rich C-tail motif on signalling.

    Article Snippet: Plasmid DNA mutations in the C-terminus of PAR2 were created using QuikChange XL Multi Site-Directed Mutagenesis kit (Agilent Technologies, Mississauga, ON, Canada) to generate all mutants described in this study.

    Techniques: Generated, Mutagenesis, Clone Assay, Construct