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    Thermo Fisher superscript vilo cdna synthesis kit thermo fisher
    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p
    Superscript Vilo Cdna Synthesis Kit Thermo Fisher, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript ii cdna synthesis kit thermo fisher scientific
    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p
    Superscript Ii Cdna Synthesis Kit Thermo Fisher Scientific, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher supersciript iii first strand synthesis kit
    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p
    Supersciript Iii First Strand Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscriptã‚â iii first strand synthesis supermix kit
    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p
    Superscriptã‚â Iii First Strand Synthesis Supermix Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript vilo kit
    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p
    Superscript Vilo Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii reverse transcriptase
    iPSC colonies formed from Aplf -kd cells express pluripotency genes. (A) C21 and C23 iPSC clones at passage 26. Scale bar: 100 µm. (B) iPSCs from colonies C21 and C23 (generated from Aplf -kd MEFs), and C3 (generated from control plko.1 MEFs) were analyzed for the expression of the pluripotency proteins OCT3/4 and NANOG by performing immunofluorescence analysis. E14 ESCs were used as control. (C) <t>qRT-PCR</t> analysis of different pluripotency genes on samples generated from different passages (p-) of E14 ESCs and of iPSCs from C21, C23 and C3. Error bars represent s.e.m. for <t>three</t> independent experiments.
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    Thermo Fisher invitrogen superscript kit
    iPSC colonies formed from Aplf -kd cells express pluripotency genes. (A) C21 and C23 iPSC clones at passage 26. Scale bar: 100 µm. (B) iPSCs from colonies C21 and C23 (generated from Aplf -kd MEFs), and C3 (generated from control plko.1 MEFs) were analyzed for the expression of the pluripotency proteins OCT3/4 and NANOG by performing immunofluorescence analysis. E14 ESCs were used as control. (C) <t>qRT-PCR</t> analysis of different pluripotency genes on samples generated from different passages (p-) of E14 ESCs and of iPSCs from C21, C23 and C3. Error bars represent s.e.m. for <t>three</t> independent experiments.
    Invitrogen Superscript Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript choice kit
    iPSC colonies formed from Aplf -kd cells express pluripotency genes. (A) C21 and C23 iPSC clones at passage 26. Scale bar: 100 µm. (B) iPSCs from colonies C21 and C23 (generated from Aplf -kd MEFs), and C3 (generated from control plko.1 MEFs) were analyzed for the expression of the pluripotency proteins OCT3/4 and NANOG by performing immunofluorescence analysis. E14 ESCs were used as control. (C) <t>qRT-PCR</t> analysis of different pluripotency genes on samples generated from different passages (p-) of E14 ESCs and of iPSCs from C21, C23 and C3. Error bars represent s.e.m. for <t>three</t> independent experiments.
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    Image Search Results


    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and cDNA were prepared using the RNesay mini kit (Qiagen) and SuperScript VILO cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p

    Journal: PLoS ONE

    Article Title: Mitochondrial DAMPs Increase Endothelial Permeability through Neutrophil Dependent and Independent Pathways

    doi: 10.1371/journal.pone.0059989

    Figure Lengend Snippet: mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and cDNA were prepared using the RNesay mini kit (Qiagen) and SuperScript VILO cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p

    Article Snippet: RNA and cDNA Preparation and Quantitative PCR RNA and cDNA were prepared using the RNesay mini kit (Qiagen, Valencia, CA) and the SuperScript VILO cDNA Synthesis kit (Life Technologies, Carlsbad, CA), respectively.

    Techniques: Expressing, Size-exclusion Chromatography, Isolation, Incubation, Real-time Polymerase Chain Reaction

    iPSC colonies formed from Aplf -kd cells express pluripotency genes. (A) C21 and C23 iPSC clones at passage 26. Scale bar: 100 µm. (B) iPSCs from colonies C21 and C23 (generated from Aplf -kd MEFs), and C3 (generated from control plko.1 MEFs) were analyzed for the expression of the pluripotency proteins OCT3/4 and NANOG by performing immunofluorescence analysis. E14 ESCs were used as control. (C) qRT-PCR analysis of different pluripotency genes on samples generated from different passages (p-) of E14 ESCs and of iPSCs from C21, C23 and C3. Error bars represent s.e.m. for three independent experiments.

    Journal: Journal of Cell Science

    Article Title: Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts

    doi: 10.1242/jcs.194035

    Figure Lengend Snippet: iPSC colonies formed from Aplf -kd cells express pluripotency genes. (A) C21 and C23 iPSC clones at passage 26. Scale bar: 100 µm. (B) iPSCs from colonies C21 and C23 (generated from Aplf -kd MEFs), and C3 (generated from control plko.1 MEFs) were analyzed for the expression of the pluripotency proteins OCT3/4 and NANOG by performing immunofluorescence analysis. E14 ESCs were used as control. (C) qRT-PCR analysis of different pluripotency genes on samples generated from different passages (p-) of E14 ESCs and of iPSCs from C21, C23 and C3. Error bars represent s.e.m. for three independent experiments.

    Article Snippet: Mouse Aplf full-length cDNA (accession number: ) was amplified from MEFs by performing PCR by using a superscript reverse transcriptase kit (Invitrogen; 18080044).

    Techniques: Clone Assay, Generated, Expressing, Immunofluorescence, Quantitative RT-PCR

    Downregulation of APLF enhances reprogramming of fibroblasts. (A) MEFs were transduced with lentiviral particles expressing shRNA against Aplf ( Aplf -shRNA) or empty plko.1 vector (empty vector control). Extent of knockdown was measured at the (A) protein and (B) mRNA level. The band intensity of blots was measured by using ImageJ software. The relative intensity of APLF to β-actin immunoreactive bands was determined, and the value for the control was normalized to 1. The intensity is the average of three independent experiments. (C) Control (with empty plko.1 vector) and Aplf- kd (expressing Aplf -shRNA) MEFs were transduced with polycistronic vector expressing OSKM under the influence of the Tet operator. Doxycycline (2 µg/ml) induced the expression of OSKM. Micrographs show iPSC colonies generated from control (left panel) and Aplf -kd MEFs (right panel). Dashed circles mark iPSC colonies. (D) Alkaline phosphatase staining of iPSC clones in control (upper panel) and Aplf -kd (lower panel) cells. Micrographs of the alkaline-phosphatase-positive clones at 10× magnification are also included. Dashed circles indicate pluripotent iPSC colonies. Similar colonies only were counted as alkaline-phosphatase-positive clones. (E) Bar graph demonstrating the number of alkaline-phosphatase-positive clones formed at day 10 and day 20 of reprogramming from control and Aplf- kd cells with respect to control set at day 10. (F) Immunofluorescence study of the expression of NANOG and OCT3/4 in control and Aplf -kd cells at day 10 of reprogramming. Error bars are s.e.m., n =3, ** P

    Journal: Journal of Cell Science

    Article Title: Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts

    doi: 10.1242/jcs.194035

    Figure Lengend Snippet: Downregulation of APLF enhances reprogramming of fibroblasts. (A) MEFs were transduced with lentiviral particles expressing shRNA against Aplf ( Aplf -shRNA) or empty plko.1 vector (empty vector control). Extent of knockdown was measured at the (A) protein and (B) mRNA level. The band intensity of blots was measured by using ImageJ software. The relative intensity of APLF to β-actin immunoreactive bands was determined, and the value for the control was normalized to 1. The intensity is the average of three independent experiments. (C) Control (with empty plko.1 vector) and Aplf- kd (expressing Aplf -shRNA) MEFs were transduced with polycistronic vector expressing OSKM under the influence of the Tet operator. Doxycycline (2 µg/ml) induced the expression of OSKM. Micrographs show iPSC colonies generated from control (left panel) and Aplf -kd MEFs (right panel). Dashed circles mark iPSC colonies. (D) Alkaline phosphatase staining of iPSC clones in control (upper panel) and Aplf -kd (lower panel) cells. Micrographs of the alkaline-phosphatase-positive clones at 10× magnification are also included. Dashed circles indicate pluripotent iPSC colonies. Similar colonies only were counted as alkaline-phosphatase-positive clones. (E) Bar graph demonstrating the number of alkaline-phosphatase-positive clones formed at day 10 and day 20 of reprogramming from control and Aplf- kd cells with respect to control set at day 10. (F) Immunofluorescence study of the expression of NANOG and OCT3/4 in control and Aplf -kd cells at day 10 of reprogramming. Error bars are s.e.m., n =3, ** P

    Article Snippet: Mouse Aplf full-length cDNA (accession number: ) was amplified from MEFs by performing PCR by using a superscript reverse transcriptase kit (Invitrogen; 18080044).

    Techniques: Transduction, Expressing, shRNA, Plasmid Preparation, Software, Generated, Staining, Clone Assay, Immunofluorescence

    APLF upregulation is associated with a decrease in expression of pluripotency factors. (A,B) MEFs and feeder-free E14 ESCs were cultured for 3 days, and mRNA and protein were extracted to determine the expression of histone chaperones by performing qRT-PCR analyses and western blot analysis. (C) E14 ESCs were differentiated in the absence of LIF and towards endodermal, mesodermal and ectodermal lineages. qRT-PCR analysis showed an increase in Aplf levels in cells that had been differentiated from E14 ESCs. (D) Full-length mouse Aplf cDNA was PCR-amplified from a cDNA library derived from mRNA isolated from MEFs and was cloned into the pUltra lentiviral vector to generate lentiviral particles to transduce E14 ESCs. Western blot analysis confirmed the ectopic overexpression of APLF in clones #8 and #10. (E) E14 ESCs with empty vector and Aplf cDNA (clone 10) were analyzed for the expression of pluripotency genes Oct3/4 and Nanog , and lineage markers Gata4 , Flk1 and nestin by performing qRT-PCR analysis. Error bars represent the s.e.m. for three independent experiments. (F) MEFs were transduced with lentiviral particles expressing OSKM and Tet to generate iPSCs. Cell lysates at different days of reprogramming were analyzed for the expression of APLF by western blotting. Error bars are s.e.m., n =3, * P

    Journal: Journal of Cell Science

    Article Title: Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts

    doi: 10.1242/jcs.194035

    Figure Lengend Snippet: APLF upregulation is associated with a decrease in expression of pluripotency factors. (A,B) MEFs and feeder-free E14 ESCs were cultured for 3 days, and mRNA and protein were extracted to determine the expression of histone chaperones by performing qRT-PCR analyses and western blot analysis. (C) E14 ESCs were differentiated in the absence of LIF and towards endodermal, mesodermal and ectodermal lineages. qRT-PCR analysis showed an increase in Aplf levels in cells that had been differentiated from E14 ESCs. (D) Full-length mouse Aplf cDNA was PCR-amplified from a cDNA library derived from mRNA isolated from MEFs and was cloned into the pUltra lentiviral vector to generate lentiviral particles to transduce E14 ESCs. Western blot analysis confirmed the ectopic overexpression of APLF in clones #8 and #10. (E) E14 ESCs with empty vector and Aplf cDNA (clone 10) were analyzed for the expression of pluripotency genes Oct3/4 and Nanog , and lineage markers Gata4 , Flk1 and nestin by performing qRT-PCR analysis. Error bars represent the s.e.m. for three independent experiments. (F) MEFs were transduced with lentiviral particles expressing OSKM and Tet to generate iPSCs. Cell lysates at different days of reprogramming were analyzed for the expression of APLF by western blotting. Error bars are s.e.m., n =3, * P

    Article Snippet: Mouse Aplf full-length cDNA (accession number: ) was amplified from MEFs by performing PCR by using a superscript reverse transcriptase kit (Invitrogen; 18080044).

    Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Derivative Assay, Isolation, Clone Assay, Plasmid Preparation, Transduction, Over Expression