plasmids ck1δtv1  (Thermo Fisher)


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    Structured Review

    Thermo Fisher plasmids ck1δtv1
    Comparison of the phosphorylation of mouse <t>CK1δTV1</t> and TV2. Purified GST-mouse CK1δTV1 and GST-mouse CK1δTV2 were autophoshorylated in vitro for 30 min. Phosphorylated proteins were separated by SDS-PAGE, blotted onto PVDF membranes, trypsinized and oxidized. The resulting phosphopeptides were separated in two dimensional analyses by electrophoresis at pH 1.9 and ascending chromatography. Phosphopeptides were labeled with capital letters . Phospopeptides H , J , K , L , and M were only present in CK1δTV1, whereas except G and I all other phosphopeptides ( A , B , C , D , E , and F ) of CK1δTV1 were reduced in comparison to those of CK1δTV2
    Plasmids Ck1δtv1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 10669 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    plasmids ck1δtv1 - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "2-Benzamido-N-(1H-benzo[d]imidazol-2-yl)thiazole-4-carboxamide derivatives as potent inhibitors of CK1?/?"

    Article Title: 2-Benzamido-N-(1H-benzo[d]imidazol-2-yl)thiazole-4-carboxamide derivatives as potent inhibitors of CK1?/?

    Journal: Amino Acids

    doi: 10.1007/s00726-012-1234-x

    Comparison of the phosphorylation of mouse CK1δTV1 and TV2. Purified GST-mouse CK1δTV1 and GST-mouse CK1δTV2 were autophoshorylated in vitro for 30 min. Phosphorylated proteins were separated by SDS-PAGE, blotted onto PVDF membranes, trypsinized and oxidized. The resulting phosphopeptides were separated in two dimensional analyses by electrophoresis at pH 1.9 and ascending chromatography. Phosphopeptides were labeled with capital letters . Phospopeptides H , J , K , L , and M were only present in CK1δTV1, whereas except G and I all other phosphopeptides ( A , B , C , D , E , and F ) of CK1δTV1 were reduced in comparison to those of CK1δTV2
    Figure Legend Snippet: Comparison of the phosphorylation of mouse CK1δTV1 and TV2. Purified GST-mouse CK1δTV1 and GST-mouse CK1δTV2 were autophoshorylated in vitro for 30 min. Phosphorylated proteins were separated by SDS-PAGE, blotted onto PVDF membranes, trypsinized and oxidized. The resulting phosphopeptides were separated in two dimensional analyses by electrophoresis at pH 1.9 and ascending chromatography. Phosphopeptides were labeled with capital letters . Phospopeptides H , J , K , L , and M were only present in CK1δTV1, whereas except G and I all other phosphopeptides ( A , B , C , D , E , and F ) of CK1δTV1 were reduced in comparison to those of CK1δTV2

    Techniques Used: Purification, In Vitro, SDS Page, Electrophoresis, Chromatography, Labeling

    The degree of phosphorylation influences the activity of CK1δ transcription variants and modulates the inhibitory effects of compound 5 . a Comparision of substrate phosphorylation of CK1δ transcription variants induced at 15°C or 37°C. Purified CK1δTV1 and CK1δTV2 either having been induced for 2 h at 37°C or for 14 h at 15°C were used for phosphorylation of GST-mouse p53 1−64 in vitro. b Determination of the inhibitory ability of compound 5 towards CK1δ transcription variants differing in their phosphorylation degree. Compound 5 was tested for its ability to inhibit CK1δTV1 and CK1δTV2 which have either been induced for 2 h at 37°C or for 14 h at 15°C. A high degree of phosphorylation of both CK1δ transcription variants resulted in recuced inhibitory effects of 5 indicated by three- to fourfold higher IC 50 values
    Figure Legend Snippet: The degree of phosphorylation influences the activity of CK1δ transcription variants and modulates the inhibitory effects of compound 5 . a Comparision of substrate phosphorylation of CK1δ transcription variants induced at 15°C or 37°C. Purified CK1δTV1 and CK1δTV2 either having been induced for 2 h at 37°C or for 14 h at 15°C were used for phosphorylation of GST-mouse p53 1−64 in vitro. b Determination of the inhibitory ability of compound 5 towards CK1δ transcription variants differing in their phosphorylation degree. Compound 5 was tested for its ability to inhibit CK1δTV1 and CK1δTV2 which have either been induced for 2 h at 37°C or for 14 h at 15°C. A high degree of phosphorylation of both CK1δ transcription variants resulted in recuced inhibitory effects of 5 indicated by three- to fourfold higher IC 50 values

    Techniques Used: Activity Assay, Purification, In Vitro

    2) Product Images from "A Novel Pax5-Binding Regulatory Element in the Ig? Locus"

    Article Title: A Novel Pax5-Binding Regulatory Element in the Ig? Locus

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2014.00240

    Effect of deletion of the Dm element at the endogenous locus on somatic hypermutations . Somatic hypermutations in B220 + PNA high Peyer’s patches germinal center B cells from (A) Igκ WT/WT , (B) Igκ ΔDm/ΔDm , and (C) Igκ WT/ΔDm mice. Pie charts indicate the number of mutations sequenced in a 188-bp region immediately downstream of the Vκ–Jκ5 joint. Number of colonies sequenced from each mouse and mutation rate per kilobase in total and mutated clones are indicated.
    Figure Legend Snippet: Effect of deletion of the Dm element at the endogenous locus on somatic hypermutations . Somatic hypermutations in B220 + PNA high Peyer’s patches germinal center B cells from (A) Igκ WT/WT , (B) Igκ ΔDm/ΔDm , and (C) Igκ WT/ΔDm mice. Pie charts indicate the number of mutations sequenced in a 188-bp region immediately downstream of the Vκ–Jκ5 joint. Number of colonies sequenced from each mouse and mutation rate per kilobase in total and mutated clones are indicated.

    Techniques Used: Mouse Assay, Mutagenesis, Clone Assay

    3) Product Images from "Identification of RNA binding motif proteins essential for cardiovascular development"

    Article Title: Identification of RNA binding motif proteins essential for cardiovascular development

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-11-62

    rbm24a and rbm24b are required for normal cardiac development . Translation blocking morpholinos complementary to rbm24a (5 ng) or rbm24b (8 ng) were injected into 1-2 cell stage zebrafish embryos and the resulting phenotypes were evaluated compared to uninjected controls at 48 hpf (A, B, D), 72 hpf (F,G,I), and 96 hpf (K, L, O). Lateral heart views are shown with a dotted outline around the heart chambers. Both morphant embryo conditions exhibited cardiac looping defects and edema at all stages. Heart chambers are shown with a dotted outline with chambers denoted: v, ventricle; a, atrium; black arrows, cardiac edema. Phenotype rescue was achieved for each rbm24 via co-injection of each respective full length capped poly-A RNA transcript ( rbm24a 800 pg, rbm24b 50 pg) along with the respective complementary translation blocking morpholino into 1-2 cell stage embryos where 800 pg rbm24a (C, H, M) or 50 pg rbm24b (E, J, P) achieved rescue. Rescued embryos posses looped hearts absent of edema.
    Figure Legend Snippet: rbm24a and rbm24b are required for normal cardiac development . Translation blocking morpholinos complementary to rbm24a (5 ng) or rbm24b (8 ng) were injected into 1-2 cell stage zebrafish embryos and the resulting phenotypes were evaluated compared to uninjected controls at 48 hpf (A, B, D), 72 hpf (F,G,I), and 96 hpf (K, L, O). Lateral heart views are shown with a dotted outline around the heart chambers. Both morphant embryo conditions exhibited cardiac looping defects and edema at all stages. Heart chambers are shown with a dotted outline with chambers denoted: v, ventricle; a, atrium; black arrows, cardiac edema. Phenotype rescue was achieved for each rbm24 via co-injection of each respective full length capped poly-A RNA transcript ( rbm24a 800 pg, rbm24b 50 pg) along with the respective complementary translation blocking morpholino into 1-2 cell stage embryos where 800 pg rbm24a (C, H, M) or 50 pg rbm24b (E, J, P) achieved rescue. Rescued embryos posses looped hearts absent of edema.

    Techniques Used: Blocking Assay, Injection

    4) Product Images from "MicroRNA-122 Triggers Mesenchymal-Epithelial Transition and Suppresses Hepatocellular Carcinoma Cell Motility and Invasion by Targeting RhoA"

    Article Title: MicroRNA-122 Triggers Mesenchymal-Epithelial Transition and Suppresses Hepatocellular Carcinoma Cell Motility and Invasion by Targeting RhoA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0101330

    A proposed model for the HNF4α/miR-122/RhoA axis in regulating EMT, and the invasion and metastasis of HCC cells.
    Figure Legend Snippet: A proposed model for the HNF4α/miR-122/RhoA axis in regulating EMT, and the invasion and metastasis of HCC cells.

    Techniques Used:

    miR-122 induced MET, and suppressed cell migration and invasion by down-regulating RhoA. ( A ) RhoA is a target gene of miR-122. The luciferase reporter assay was performed using Bel-7402 and 293T cells as described in the Materials and methods section. ( B ) miR-122 triggered MET-like cellular marker alterations through the down-regulation of RhoA. Western blot analysis comparing the expression of epithelial markers (ZO-1 and α-catenin) and mesenchymal markers (vimentin and fibronectin) in Bel-7402 cells expressing the control vector, miR-122, miR-122/RhoA-wt, and RhoA-T19N. ( C ) miR-122 inhibited cell migration and invasion in vitro through RhoA targeting. The effects of miR-122, miR-122/RhoA-wt, and RhoA-T19N on cell migration and invasion were evaluated using transwell and Matrigel-coated Boyden chambers, respectively.
    Figure Legend Snippet: miR-122 induced MET, and suppressed cell migration and invasion by down-regulating RhoA. ( A ) RhoA is a target gene of miR-122. The luciferase reporter assay was performed using Bel-7402 and 293T cells as described in the Materials and methods section. ( B ) miR-122 triggered MET-like cellular marker alterations through the down-regulation of RhoA. Western blot analysis comparing the expression of epithelial markers (ZO-1 and α-catenin) and mesenchymal markers (vimentin and fibronectin) in Bel-7402 cells expressing the control vector, miR-122, miR-122/RhoA-wt, and RhoA-T19N. ( C ) miR-122 inhibited cell migration and invasion in vitro through RhoA targeting. The effects of miR-122, miR-122/RhoA-wt, and RhoA-T19N on cell migration and invasion were evaluated using transwell and Matrigel-coated Boyden chambers, respectively.

    Techniques Used: Migration, Luciferase, Reporter Assay, Marker, Western Blot, Expressing, Plasmid Preparation, In Vitro

    5) Product Images from "Expression of C-terminal deleted p53 isoforms in neuroblastoma"

    Article Title: Expression of C-terminal deleted p53 isoforms in neuroblastoma

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl619

    p53 transactivation ability by luciferase test using plasmid pE1B-hWAF1( A ) and Bax ( B ). p53-deficient LAN-1 cells (p53-) (left panel) or SH-SY5Y (p53+) (right panel) were cotransfected with 0.5 μg of the luciferase reporter gene containing the human p21/WAF1 -p53-responsive element and with 1 μg of the expressing vector as indicated. Cells were collected and subject to luciferase assay, 24 h following cotransfection. The values represent mean relative luciferase activity from three independent experiments. SH-SY5Y, IGR-N-91, SK-N-BE(2), SK-N-AS and IGR-NB8 were termed as SH, N91, BE(2), AS and NB8, respectively.
    Figure Legend Snippet: p53 transactivation ability by luciferase test using plasmid pE1B-hWAF1( A ) and Bax ( B ). p53-deficient LAN-1 cells (p53-) (left panel) or SH-SY5Y (p53+) (right panel) were cotransfected with 0.5 μg of the luciferase reporter gene containing the human p21/WAF1 -p53-responsive element and with 1 μg of the expressing vector as indicated. Cells were collected and subject to luciferase assay, 24 h following cotransfection. The values represent mean relative luciferase activity from three independent experiments. SH-SY5Y, IGR-N-91, SK-N-BE(2), SK-N-AS and IGR-NB8 were termed as SH, N91, BE(2), AS and NB8, respectively.

    Techniques Used: Luciferase, Plasmid Preparation, Expressing, Cotransfection, Activity Assay

    Structure of p53 proteins in different neuroblastoma cell lines. The three functional domains are represented: TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. The wild-type p53 gene in SH-SY5Y, IMR-32 and LAN-5 cells contains 11 exons that encode 393 amino acids. In SK-N-BE(2) cells, p53 is mutated at codon 135 ( * ), which converts cysteine to phenylalanine. In IGR-N-91 cells, a duplication of exons 7-8-9 adds an additional 107 amino acids leading to a total of 500. In SK-N-AS cells, a mutation due to alternate splicing downstream of exon 9 leads to a protein of 341 amino acids whereas in IGR-NB8 cells, the p53 protein ends at 326 amino acids owing to the mutation E326STOP.
    Figure Legend Snippet: Structure of p53 proteins in different neuroblastoma cell lines. The three functional domains are represented: TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. The wild-type p53 gene in SH-SY5Y, IMR-32 and LAN-5 cells contains 11 exons that encode 393 amino acids. In SK-N-BE(2) cells, p53 is mutated at codon 135 ( * ), which converts cysteine to phenylalanine. In IGR-N-91 cells, a duplication of exons 7-8-9 adds an additional 107 amino acids leading to a total of 500. In SK-N-AS cells, a mutation due to alternate splicing downstream of exon 9 leads to a protein of 341 amino acids whereas in IGR-NB8 cells, the p53 protein ends at 326 amino acids owing to the mutation E326STOP.

    Techniques Used: Functional Assay, Binding Assay, Mutagenesis

    6) Product Images from "Increased expression of transcription factor TFAP2? correlates with chemosensitivity in advanced bladder cancer"

    Article Title: Increased expression of transcription factor TFAP2? correlates with chemosensitivity in advanced bladder cancer

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-11-135

    Expression of TFAP2α isoforms . (A) Expression of TFAP2α isoform 1, 2 and 3 in advanced muscle invasive bladder cancer (T2-4) were determined using real-time Q-PCR. Analysis was performed on cDNA from 10 tumor specimens and each bar represents the mean from the 10 samples.(B) COS-7 cells were transiently transfected with empty pcDNA3.1/V5-His vector (lane 2, 6), pcDNA3.1/V5-His- TFAP2α isoform 1(lane 3, 7), isoform 2 (lane 4, 8) and isoform 3 (lane 5, 9). Western blot of 30 μg total protein lysate from non-transfected HU609 bladder cells (lane 1) and COS-7 transfected cells (lane 2-9) 48 h post transfection probed with anti TFAP2α antibody (lane 1-5) or anti-V5 antibody (lane 6-9).
    Figure Legend Snippet: Expression of TFAP2α isoforms . (A) Expression of TFAP2α isoform 1, 2 and 3 in advanced muscle invasive bladder cancer (T2-4) were determined using real-time Q-PCR. Analysis was performed on cDNA from 10 tumor specimens and each bar represents the mean from the 10 samples.(B) COS-7 cells were transiently transfected with empty pcDNA3.1/V5-His vector (lane 2, 6), pcDNA3.1/V5-His- TFAP2α isoform 1(lane 3, 7), isoform 2 (lane 4, 8) and isoform 3 (lane 5, 9). Western blot of 30 μg total protein lysate from non-transfected HU609 bladder cells (lane 1) and COS-7 transfected cells (lane 2-9) 48 h post transfection probed with anti TFAP2α antibody (lane 1-5) or anti-V5 antibody (lane 6-9).

    Techniques Used: Expressing, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot

    7) Product Images from "Decreased Striatal RGS2 Expression Is Neuroprotective in Huntington's Disease (HD) and Exemplifies a Compensatory Aspect of HD-Induced Gene Regulation"

    Article Title: Decreased Striatal RGS2 Expression Is Neuroprotective in Huntington's Disease (HD) and Exemplifies a Compensatory Aspect of HD-Induced Gene Regulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022231

    Silencing of RGS2 or RASD2 is protective in the TRE-htt171-82Q primary striatal neuron model of HD. A RGS2-targeting shRNA delivered via a lentiviral vector resulted in a 46.1±7.2% reduction of RGS2 mRNA (in neurons) and 40±5.4% reduction of RGS2 protein expression (in HEK 293T cells), respectively (Panel A). A RASD2-targeting shRNA resulted in a 76.8±0.3% reduction of RASD2 mRNA expression (in neurons) and 79.1±3.6% reduction of RASD2 protein expression (in HEK 293T cells), respectively (Panel B). Immunoblots were performed on HEK 293 cells expressing HA-tagged RGS2 and RGS2 silencing constructs. Lentiviral delivery of an shRNA targeting GFP (shGFP) was used as a control. Values are presented as mean±SEM, n = 3. * p
    Figure Legend Snippet: Silencing of RGS2 or RASD2 is protective in the TRE-htt171-82Q primary striatal neuron model of HD. A RGS2-targeting shRNA delivered via a lentiviral vector resulted in a 46.1±7.2% reduction of RGS2 mRNA (in neurons) and 40±5.4% reduction of RGS2 protein expression (in HEK 293T cells), respectively (Panel A). A RASD2-targeting shRNA resulted in a 76.8±0.3% reduction of RASD2 mRNA expression (in neurons) and 79.1±3.6% reduction of RASD2 protein expression (in HEK 293T cells), respectively (Panel B). Immunoblots were performed on HEK 293 cells expressing HA-tagged RGS2 and RGS2 silencing constructs. Lentiviral delivery of an shRNA targeting GFP (shGFP) was used as a control. Values are presented as mean±SEM, n = 3. * p

    Techniques Used: shRNA, Plasmid Preparation, Expressing, Western Blot, Construct

    Modulation of RGS2 expression does not influence nuclear accumulation of mutant htt. Primary striatal neurons were infected on DIV1 with lentiviral constructs encoding WT (18Q) or mutant (82Q) htt171 fragments under the control of a TRE promoter and co-infected on DIV4 with an RGS2 expression vector as described in Fig. 2B (A) or RGS2 silencing construct as in Fig. 4B (B). NeuN-positive and 2B4-positive nuclei were quantified as described in the Methods section. (C) Top: representative images of NeuN and 2B4 staining; scale bar 100 µm. Bottom: Representative higher magnification images showing the continued presence of 2B4-positive nuclei after RGS2 silencing; scale bar = 10 µm.
    Figure Legend Snippet: Modulation of RGS2 expression does not influence nuclear accumulation of mutant htt. Primary striatal neurons were infected on DIV1 with lentiviral constructs encoding WT (18Q) or mutant (82Q) htt171 fragments under the control of a TRE promoter and co-infected on DIV4 with an RGS2 expression vector as described in Fig. 2B (A) or RGS2 silencing construct as in Fig. 4B (B). NeuN-positive and 2B4-positive nuclei were quantified as described in the Methods section. (C) Top: representative images of NeuN and 2B4 staining; scale bar 100 µm. Bottom: Representative higher magnification images showing the continued presence of 2B4-positive nuclei after RGS2 silencing; scale bar = 10 µm.

    Techniques Used: Expressing, Mutagenesis, Infection, Construct, Plasmid Preparation, Staining

    RGS2 levels modulate 3NP toxicity. Primary striatal neurons were infected on DIV 4 with an RGS2 lentiviral expression vector at a concentration of 8 ng/ml p24 (A) or a lentiviral silencing construct targeting RGS2 at concentration of 1 ng/ml p24 (B). Vectors encoding CFP or shGFP were used as controls (in A and B, respectively). After 2 weeks in culture, neurons were treated with 100 or 300 uM 3NP. Neuronal viability was assessed after 48 h by NeuN counting.
    Figure Legend Snippet: RGS2 levels modulate 3NP toxicity. Primary striatal neurons were infected on DIV 4 with an RGS2 lentiviral expression vector at a concentration of 8 ng/ml p24 (A) or a lentiviral silencing construct targeting RGS2 at concentration of 1 ng/ml p24 (B). Vectors encoding CFP or shGFP were used as controls (in A and B, respectively). After 2 weeks in culture, neurons were treated with 100 or 300 uM 3NP. Neuronal viability was assessed after 48 h by NeuN counting.

    Techniques Used: Infection, Expressing, Plasmid Preparation, Concentration Assay, Construct

    Effects of restoring downregulated RNAs in the TRE-htt171-82Q primary striatal model of HD. Primary striatal neurons were infected on DIV1 with lentiviral vectors encoding WT (18Q) or mutant (82Q) htt171 fragments under the control of a TRE promoter and co-infected on DIV4 with vectors encoding RGS2, RASD2, NNAT, STEP or CFP (control) at a concentration of 25 ng p24 antigen/ml. Neuronal viability was assessed after 3 weeks in culture by quantification of NeuN-positive cells. * indicates significance of mutant htt effects; + indicates significance of effects of the candidate modifier expression. Data are presented as mean±SEM, n = 15, expressed as % control (relative to WT htt171-18Q+CFP). * p
    Figure Legend Snippet: Effects of restoring downregulated RNAs in the TRE-htt171-82Q primary striatal model of HD. Primary striatal neurons were infected on DIV1 with lentiviral vectors encoding WT (18Q) or mutant (82Q) htt171 fragments under the control of a TRE promoter and co-infected on DIV4 with vectors encoding RGS2, RASD2, NNAT, STEP or CFP (control) at a concentration of 25 ng p24 antigen/ml. Neuronal viability was assessed after 3 weeks in culture by quantification of NeuN-positive cells. * indicates significance of mutant htt effects; + indicates significance of effects of the candidate modifier expression. Data are presented as mean±SEM, n = 15, expressed as % control (relative to WT htt171-18Q+CFP). * p

    Techniques Used: Infection, Mutagenesis, Concentration Assay, Expressing

    Effects of candidate modifiers at lower expression levels in the TRE-htt171-82Q primary striatal neuron model of HD. Primary striatal neurons were infected on DIV1 with lentiviral constructs encoding WT (18Q) or mutant (82Q) htt171 fragments under the control of a TRE promoter and co-infected on DIV4 with constructs encoding RGS2, RASD2, or CFP (control) at the concentration of 8 or 2.5 ng p24 antigen/ml. Neuronal viability was assessed after 3w in culture by quantification of NeuN-positive cells. * indicates significance of mutant htt effects; + indicates significance of effects of candidate modifier expression. Values are presented as mean±SEM, n = 15, expressed as % control (WT htt171-18Q+CFP). * p
    Figure Legend Snippet: Effects of candidate modifiers at lower expression levels in the TRE-htt171-82Q primary striatal neuron model of HD. Primary striatal neurons were infected on DIV1 with lentiviral constructs encoding WT (18Q) or mutant (82Q) htt171 fragments under the control of a TRE promoter and co-infected on DIV4 with constructs encoding RGS2, RASD2, or CFP (control) at the concentration of 8 or 2.5 ng p24 antigen/ml. Neuronal viability was assessed after 3w in culture by quantification of NeuN-positive cells. * indicates significance of mutant htt effects; + indicates significance of effects of candidate modifier expression. Values are presented as mean±SEM, n = 15, expressed as % control (WT htt171-18Q+CFP). * p

    Techniques Used: Expressing, Infection, Construct, Mutagenesis, Concentration Assay

    Timecourse of differential expression in the TRE-htt171-82Q primary striatal lentiviral model of HD. RGS2, RASD2, NNAT and STEP RNA levels were measured by qPCR in primary striatal cells expressing mutant htt fragments under the control of a TRE promoter. Target RNA levels (i.e those of RGS2, RASD2, NNAT, STEP) were normalized to the expression level of β-actin and expressed as % control relative to neurons expressing htt171-18Q at the corresponding time point. (mean±SEM); n = 4. * p
    Figure Legend Snippet: Timecourse of differential expression in the TRE-htt171-82Q primary striatal lentiviral model of HD. RGS2, RASD2, NNAT and STEP RNA levels were measured by qPCR in primary striatal cells expressing mutant htt fragments under the control of a TRE promoter. Target RNA levels (i.e those of RGS2, RASD2, NNAT, STEP) were normalized to the expression level of β-actin and expressed as % control relative to neurons expressing htt171-18Q at the corresponding time point. (mean±SEM); n = 4. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mutagenesis

    8) Product Images from "Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment"

    Article Title: Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00600-2

    Pair-wise mixed culture competition experiments. Competence assays of environmental strains Pseudomonas sp. 1SL5 and E . coli 4IgSN1, against the corresponding ARGC ( aadB or bla VIM-2 ) inserted at the native attI1 site containing strain were conducted in order to determine the relative fitness. Error bars indicate 95% confidence intervals. By definition, a relative fitness (w) of 1.00 indicates no difference in relative fitness. Relative fitness values: Pseudomonas sp. 1SL5:: aadB W = 1.03 (0.88–1.18), E . coli 4IgSN1:: aadB W = 1.02 (0.86–1.17), E . coli 4IgSN1:: bla VIM-2 W = 0.97 (0.92–1.01). CI is confidence interval.
    Figure Legend Snippet: Pair-wise mixed culture competition experiments. Competence assays of environmental strains Pseudomonas sp. 1SL5 and E . coli 4IgSN1, against the corresponding ARGC ( aadB or bla VIM-2 ) inserted at the native attI1 site containing strain were conducted in order to determine the relative fitness. Error bars indicate 95% confidence intervals. By definition, a relative fitness (w) of 1.00 indicates no difference in relative fitness. Relative fitness values: Pseudomonas sp. 1SL5:: aadB W = 1.03 (0.88–1.18), E . coli 4IgSN1:: aadB W = 1.02 (0.86–1.17), E . coli 4IgSN1:: bla VIM-2 W = 0.97 (0.92–1.01). CI is confidence interval.

    Techniques Used: Bla VIM Assay

    Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.
    Figure Legend Snippet: Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.

    Techniques Used: Isolation, Modification, Selection, Transformation Assay, Bla VIM Assay, Functional Assay, Plasmid Preparation

    9) Product Images from "Enzymatic engineering of the porcine genome with transposons and recombinases"

    Article Title: Enzymatic engineering of the porcine genome with transposons and recombinases

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-7-42

    Conditional gene-activation of integrated transposons . Colonies from the transfection of pTC-loxPTK-G with pKUb-SB11 (Fig 5C) were expanded in selective media containing puromycin. DNA from these transgenic colonies was isolated and analyzed by Southern hybridization. A) A schematic of pKT2C-loxPTK-G that shows the AseI restriction sites and the location of the PTK hybridization probe (diagonal lined rectangle) used for Southern analysis. B) A Southern blot of pKT2C-loxPTK-G colonies. The clones were analyzed without Cre excision, so integrants that result from transposition should be equal to or greater than the transposon size of 4.9 kb (open arrow). Whereas, bands associated with concatemer formation are found at 6.0 kb (vertical line arrow). The positions of the DNA marker bands of the 1 kb Quanti-Marker from ISC Bioexpress (Kaysville, Utah), are indicated by black dots on the right of each blot with sizes of 12, 10, 8, 6, 5, 4, 3, 2.5, and 2 kb shown. C) pKT2C-loxPTK-G colonies were transfected with pPGK-nlsCre and plated under gancyclovir selection. Clones with PTK eliminated by recombination became gancyclovir resistant and were counted. Cre-activation of all clones was determined to be significant (p
    Figure Legend Snippet: Conditional gene-activation of integrated transposons . Colonies from the transfection of pTC-loxPTK-G with pKUb-SB11 (Fig 5C) were expanded in selective media containing puromycin. DNA from these transgenic colonies was isolated and analyzed by Southern hybridization. A) A schematic of pKT2C-loxPTK-G that shows the AseI restriction sites and the location of the PTK hybridization probe (diagonal lined rectangle) used for Southern analysis. B) A Southern blot of pKT2C-loxPTK-G colonies. The clones were analyzed without Cre excision, so integrants that result from transposition should be equal to or greater than the transposon size of 4.9 kb (open arrow). Whereas, bands associated with concatemer formation are found at 6.0 kb (vertical line arrow). The positions of the DNA marker bands of the 1 kb Quanti-Marker from ISC Bioexpress (Kaysville, Utah), are indicated by black dots on the right of each blot with sizes of 12, 10, 8, 6, 5, 4, 3, 2.5, and 2 kb shown. C) pKT2C-loxPTK-G colonies were transfected with pPGK-nlsCre and plated under gancyclovir selection. Clones with PTK eliminated by recombination became gancyclovir resistant and were counted. Cre-activation of all clones was determined to be significant (p

    Techniques Used: Activation Assay, Transfection, Transgenic Assay, Isolation, Hybridization, Southern Blot, Clone Assay, Marker, Selection

    A CRE-Activated Transgene . A) An illustration of the Cre-activated transgene vector. The full vector, pTC-loxPTK-G, produces PTK from the mini-CAGs promoter. Transcriptional leakage into the downstream gene, GFP, is limited due to the incorporation of three full poly-adenylation signals, a so-called triple-stop. Recombination by Cre eliminates PTK and triple-stop, activating GFP expression from pTC-lox-G. B) pTC-loxPTK-G was transfected into PEGE cells with (+Cre) or without (-Cre) pPGK-nlsCre. Cells were monitored for GFP expression by fluorescent microscopy (image inserts) and flow cytometry. The percentage of cells expressing GFP was dependent on co-transfection with pPGK-nlsCre. C) PEGE cells were transfected with pTC-loxPTK-G along with pPGK-nlsCre (+Cre), pCMV-β (+βgal), pKUb-SB11 (+SB), or pKUb-SB11 and pPGK-nlsCre (+SB +Cre). The cells were plated in puromycin selective media and colonies were counted.
    Figure Legend Snippet: A CRE-Activated Transgene . A) An illustration of the Cre-activated transgene vector. The full vector, pTC-loxPTK-G, produces PTK from the mini-CAGs promoter. Transcriptional leakage into the downstream gene, GFP, is limited due to the incorporation of three full poly-adenylation signals, a so-called triple-stop. Recombination by Cre eliminates PTK and triple-stop, activating GFP expression from pTC-lox-G. B) pTC-loxPTK-G was transfected into PEGE cells with (+Cre) or without (-Cre) pPGK-nlsCre. Cells were monitored for GFP expression by fluorescent microscopy (image inserts) and flow cytometry. The percentage of cells expressing GFP was dependent on co-transfection with pPGK-nlsCre. C) PEGE cells were transfected with pTC-loxPTK-G along with pPGK-nlsCre (+Cre), pCMV-β (+βgal), pKUb-SB11 (+SB), or pKUb-SB11 and pPGK-nlsCre (+SB +Cre). The cells were plated in puromycin selective media and colonies were counted.

    Techniques Used: Plasmid Preparation, Expressing, Transfection, Microscopy, Flow Cytometry, Cytometry, Cotransfection

    Sleeping Beauty function in pig cells . A) Diagrams of the DNA vectors transfected into pig cells. pT2-FloxP-PTK is the experimental SB transposon. The transposon is flanked by inverted terminal repeats (ITR). The puromycin phosphotransferase-thymidine kinase fusion protein (PTK) is flanked by recombinase recognition sites, FRT and loxP , for Flp and Cre, respectively. pKUb-SB11 is the source of transposase and is expressed from the ubiquitin promoter (Ub). pKUb-SBΔDDE is a non-functional version of transposase because of an internal deletion within the catalytic domain. pCMV-β functions as negative control. B) The colony forming ability of pT2-FloxP-PTK in pig fetal fibroblast (PFF) and porcine endometrial gland epithelium (PEGE) was determined by counting puromycin resistant colonies after plating 60,000 cells on 10 cm dishes when pT2-FloxP-PTK was co-transfected with pKUb-SB11 (+SB), pKUb-SBΔDDE (+SBΔ DDE ), or pCMV-β (+βgal). The addition of functional transposase (+SB) versus a non-functional transposase (SBΔDDE) or pCMV-β (Bgal) was determined to be significant by analysis with an unpaired t-test (p-values
    Figure Legend Snippet: Sleeping Beauty function in pig cells . A) Diagrams of the DNA vectors transfected into pig cells. pT2-FloxP-PTK is the experimental SB transposon. The transposon is flanked by inverted terminal repeats (ITR). The puromycin phosphotransferase-thymidine kinase fusion protein (PTK) is flanked by recombinase recognition sites, FRT and loxP , for Flp and Cre, respectively. pKUb-SB11 is the source of transposase and is expressed from the ubiquitin promoter (Ub). pKUb-SBΔDDE is a non-functional version of transposase because of an internal deletion within the catalytic domain. pCMV-β functions as negative control. B) The colony forming ability of pT2-FloxP-PTK in pig fetal fibroblast (PFF) and porcine endometrial gland epithelium (PEGE) was determined by counting puromycin resistant colonies after plating 60,000 cells on 10 cm dishes when pT2-FloxP-PTK was co-transfected with pKUb-SB11 (+SB), pKUb-SBΔDDE (+SBΔ DDE ), or pCMV-β (+βgal). The addition of functional transposase (+SB) versus a non-functional transposase (SBΔDDE) or pCMV-β (Bgal) was determined to be significant by analysis with an unpaired t-test (p-values

    Techniques Used: Transfection, Functional Assay, Negative Control

    10) Product Images from "Alternative splicing, phylogenetic analysis, and craniofacial expression of zebrafish tbx22"

    Article Title: Alternative splicing, phylogenetic analysis, and craniofacial expression of zebrafish tbx22

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    doi: 10.1002/dvdy.21962

    Developmental RT-PCR analysis of tbx22-1 and tbx22-2 mRNAs
    Figure Legend Snippet: Developmental RT-PCR analysis of tbx22-1 and tbx22-2 mRNAs

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Detailed genomic organization of zebrafish tbx22-1 and tbx22-2
    Figure Legend Snippet: Detailed genomic organization of zebrafish tbx22-1 and tbx22-2

    Techniques Used:

    Alternatively spliced zebrafish tbx22 transcripts, tbx22-1 and tbx22-2
    Figure Legend Snippet: Alternatively spliced zebrafish tbx22 transcripts, tbx22-1 and tbx22-2

    Techniques Used:

    11) Product Images from "A Novel Protein, TtpC, Is a Required Component of the TonB2 Complex for Specific Iron Transport in the Pathogens Vibrio anguillarum and Vibrio cholerae ▿"

    Article Title: A Novel Protein, TtpC, Is a Required Component of the TonB2 Complex for Specific Iron Transport in the Pathogens Vibrio anguillarum and Vibrio cholerae ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00451-06

    Western blot analysis of TonB2 of V. anguillarum expressed in V. cholerae . Lane 1, V. cholerae DOV221 expressing TonB2 from V. anguillarum from plasmid pMS789; lane 2, wild-type V. cholerae CA401.
    Figure Legend Snippet: Western blot analysis of TonB2 of V. anguillarum expressed in V. cholerae . Lane 1, V. cholerae DOV221 expressing TonB2 from V. anguillarum from plasmid pMS789; lane 2, wild-type V. cholerae CA401.

    Techniques Used: Western Blot, Expressing, Plasmid Preparation

    12) Product Images from "Polyadenylation proteins CstF-64 and ?CstF-64 exhibit differential binding affinities for RNA polymers"

    Article Title: Polyadenylation proteins CstF-64 and ?CstF-64 exhibit differential binding affinities for RNA polymers

    Journal: Biochemical Journal

    doi: 10.1042/BJ20061097

    Competition/UV-cross-linking assay to determine relative affinities of RNA binding to the CstF-64 or τCstF-64 RBDs ( A ) Representative Dixon plot showing inhibition of UV cross-linking of the GST–CstF-64 RBD to 6.8 and 17 pmol of [ 32 P]poly(U) 18 by unlabelled poly(U) 18 . See the Experimental section for details. ( B ) Representative autoradiograph of GST–CstF-64 RBD binding to [ 32 P]poly(U) 18 (17 pmol) in the presence of increasing amounts (in pmol as indicated) of unlabelled poly(U) 18 . The arrow indicates the approximate mobility of the GST–CstF-64 RBD construct as determined by Coomassie Blue staining.
    Figure Legend Snippet: Competition/UV-cross-linking assay to determine relative affinities of RNA binding to the CstF-64 or τCstF-64 RBDs ( A ) Representative Dixon plot showing inhibition of UV cross-linking of the GST–CstF-64 RBD to 6.8 and 17 pmol of [ 32 P]poly(U) 18 by unlabelled poly(U) 18 . See the Experimental section for details. ( B ) Representative autoradiograph of GST–CstF-64 RBD binding to [ 32 P]poly(U) 18 (17 pmol) in the presence of increasing amounts (in pmol as indicated) of unlabelled poly(U) 18 . The arrow indicates the approximate mobility of the GST–CstF-64 RBD construct as determined by Coomassie Blue staining.

    Techniques Used: RNA Binding Assay, Inhibition, Autoradiography, Binding Assay, Construct, Staining

    Conditions of UV cross-linking and RNA binding ( A ) GST–CstF-64 RBD was bound to 32 ], exposed to increasing amounts of UV light from 0 J/cm 2 (lane 1) to 2.5 J/cm 2 (lanes 2–6), and separated by SDS/10% PAGE, dried, and analysed by autoradiography. Arrows indicate the mobility of GST–CstF-64 RBD (∼45 kDa) and free RNA. ( B ) Increasing amounts of [ 32 P]poly(U) 18 from 0.32 to 320 fmol was incubated with 0.2 μg of either GST–CstF-64 RBD (lanes 1–4), GST–τCstF-64 RBD (lanes 5–8), or GST (lane 9). After a 30 min incubation, the material was irradiated with 1.0 J/cm 2 of UV light and processed for analysis as indicated in the Experimental section. Arrows indicate the mobilities of GST–CstF-64 RBD, GST–τCstF-64 RBD (∼45 kDa) and GST (∼26 kDa). ( C ) Coomassie Blue staining of SDS/PAGE from ( B ), indicating mobilities of GST–CstF-64 RBD, GST–τCstF-64 RBD and GST. Note that the Coomassie-stained product at approx. 26 kDa in lanes 1–8 is a breakdown product of the GST-fusion proteins.
    Figure Legend Snippet: Conditions of UV cross-linking and RNA binding ( A ) GST–CstF-64 RBD was bound to 32 ], exposed to increasing amounts of UV light from 0 J/cm 2 (lane 1) to 2.5 J/cm 2 (lanes 2–6), and separated by SDS/10% PAGE, dried, and analysed by autoradiography. Arrows indicate the mobility of GST–CstF-64 RBD (∼45 kDa) and free RNA. ( B ) Increasing amounts of [ 32 P]poly(U) 18 from 0.32 to 320 fmol was incubated with 0.2 μg of either GST–CstF-64 RBD (lanes 1–4), GST–τCstF-64 RBD (lanes 5–8), or GST (lane 9). After a 30 min incubation, the material was irradiated with 1.0 J/cm 2 of UV light and processed for analysis as indicated in the Experimental section. Arrows indicate the mobilities of GST–CstF-64 RBD, GST–τCstF-64 RBD (∼45 kDa) and GST (∼26 kDa). ( C ) Coomassie Blue staining of SDS/PAGE from ( B ), indicating mobilities of GST–CstF-64 RBD, GST–τCstF-64 RBD and GST. Note that the Coomassie-stained product at approx. 26 kDa in lanes 1–8 is a breakdown product of the GST-fusion proteins.

    Techniques Used: RNA Binding Assay, Polyacrylamide Gel Electrophoresis, Autoradiography, Incubation, Irradiation, Staining, SDS Page

    Contributions of different amino acids to CstF-64 and τCstF-64 binding preferences to poly(U) ( A , B ) K d values of GST–CstF-64 RBD and mutants for poly(U) 18 . ( A ) Amino acid 41 does not contribute to poly(U) 18 RNA-binding specificity. When the GST–CstF-64 RBD construct is mutated at amino acid 41 from a proline residue to a serine residue (CstF-64 P41S), there is no change in binding towards poly(U) 18 ( K d =0.64±0.45 μM; no statistical difference is noted according to ANOVA analysis). ( B ) Amino acids downstream of the C-terminal α-helix contribute to RNA-binding specificity. When the GST–CstF-64 RBD construct was mutated at nine amino acids C-terminal to α 3 -helix (CstF-64 9AA), there was a decrease in binding to poly(U) 18 ( K d =1.31±0.29 μM). This changes the binding of CstF-64 RBD to resemble that of τCstF-64 for poly(U) 18 .
    Figure Legend Snippet: Contributions of different amino acids to CstF-64 and τCstF-64 binding preferences to poly(U) ( A , B ) K d values of GST–CstF-64 RBD and mutants for poly(U) 18 . ( A ) Amino acid 41 does not contribute to poly(U) 18 RNA-binding specificity. When the GST–CstF-64 RBD construct is mutated at amino acid 41 from a proline residue to a serine residue (CstF-64 P41S), there is no change in binding towards poly(U) 18 ( K d =0.64±0.45 μM; no statistical difference is noted according to ANOVA analysis). ( B ) Amino acids downstream of the C-terminal α-helix contribute to RNA-binding specificity. When the GST–CstF-64 RBD construct was mutated at nine amino acids C-terminal to α 3 -helix (CstF-64 9AA), there was a decrease in binding to poly(U) 18 ( K d =1.31±0.29 μM). This changes the binding of CstF-64 RBD to resemble that of τCstF-64 for poly(U) 18 .

    Techniques Used: Binding Assay, RNA Binding Assay, Construct

    RNA-binding affinities of CstF-64 and τCstF-64 for ribopolymers [poly(U) 18 , poly(A) 18 , poly(G) 18 , poly(C) 18 and poly(GU) 9 ] ( A ) K d values (micromolar) relative to poly(U) 18 of the GST–CstF-64 RBD for poly(U) 18 ( K d =0.66±0.13 μM), poly(A) 18 ( K d =5.4±2.05 μM), poly(G) 18 ( K d =6.51±0.74 μM), poly(C) 18 ( K d =18.44±1.62 μM) or poly(GU) 9 ( K d =3.09±0.74 μM). Poly(U) 18 ( P
    Figure Legend Snippet: RNA-binding affinities of CstF-64 and τCstF-64 for ribopolymers [poly(U) 18 , poly(A) 18 , poly(G) 18 , poly(C) 18 and poly(GU) 9 ] ( A ) K d values (micromolar) relative to poly(U) 18 of the GST–CstF-64 RBD for poly(U) 18 ( K d =0.66±0.13 μM), poly(A) 18 ( K d =5.4±2.05 μM), poly(G) 18 ( K d =6.51±0.74 μM), poly(C) 18 ( K d =18.44±1.62 μM) or poly(GU) 9 ( K d =3.09±0.74 μM). Poly(U) 18 ( P

    Techniques Used: RNA Binding Assay

    13) Product Images from "Expression of C-terminal deleted p53 isoforms in neuroblastoma"

    Article Title: Expression of C-terminal deleted p53 isoforms in neuroblastoma

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl619

    p53 transactivation ability using yeast-based assay (FASAY). ( A ) Schematic representation of the analysis of p53 mutants using the yeast homologous recombination expression vector pRDI-22 carrying the 5′ and 3′ ends of the p53 open reading frame and the split forms, pFW35 and pFW34 (lacking p53 fragment from amino acids 66 to 210 for split 5′ and 211–348 for split 3′, respectively) transfected into YPH500 Ade2 yeast strain. This strain repairs double-strand breaks in transfected plasmids by homologous recombination as ‘gap repair’ (see text). ( B ) Photographs of yeast colonies showing 100% wild-type p53 where all colonies are white (a), or special mutated p53 by duplication of exons 7–9 found in IGR-N-91 cells (b), where white and red colonies were mixed (see also Table 2 ), and mutated p53 such as those in SK-N-BE(2), where all colonies are red (c).
    Figure Legend Snippet: p53 transactivation ability using yeast-based assay (FASAY). ( A ) Schematic representation of the analysis of p53 mutants using the yeast homologous recombination expression vector pRDI-22 carrying the 5′ and 3′ ends of the p53 open reading frame and the split forms, pFW35 and pFW34 (lacking p53 fragment from amino acids 66 to 210 for split 5′ and 211–348 for split 3′, respectively) transfected into YPH500 Ade2 yeast strain. This strain repairs double-strand breaks in transfected plasmids by homologous recombination as ‘gap repair’ (see text). ( B ) Photographs of yeast colonies showing 100% wild-type p53 where all colonies are white (a), or special mutated p53 by duplication of exons 7–9 found in IGR-N-91 cells (b), where white and red colonies were mixed (see also Table 2 ), and mutated p53 such as those in SK-N-BE(2), where all colonies are red (c).

    Techniques Used: Homologous Recombination, Expressing, Plasmid Preparation, Transfection

    p53 transactivation ability by luciferase test using plasmid pE1B-hWAF1( A ) and Bax ( B ). p53-deficient LAN-1 cells (p53-) (left panel) or SH-SY5Y (p53+) (right panel) were cotransfected with 0.5 μg of the luciferase reporter gene containing the human p21/WAF1 -p53-responsive element and with 1 μg of the expressing vector as indicated. Cells were collected and subject to luciferase assay, 24 h following cotransfection. The values represent mean relative luciferase activity from three independent experiments. SH-SY5Y, IGR-N-91, SK-N-BE(2), SK-N-AS and IGR-NB8 were termed as SH, N91, BE(2), AS and NB8, respectively.
    Figure Legend Snippet: p53 transactivation ability by luciferase test using plasmid pE1B-hWAF1( A ) and Bax ( B ). p53-deficient LAN-1 cells (p53-) (left panel) or SH-SY5Y (p53+) (right panel) were cotransfected with 0.5 μg of the luciferase reporter gene containing the human p21/WAF1 -p53-responsive element and with 1 μg of the expressing vector as indicated. Cells were collected and subject to luciferase assay, 24 h following cotransfection. The values represent mean relative luciferase activity from three independent experiments. SH-SY5Y, IGR-N-91, SK-N-BE(2), SK-N-AS and IGR-NB8 were termed as SH, N91, BE(2), AS and NB8, respectively.

    Techniques Used: Luciferase, Plasmid Preparation, Expressing, Cotransfection, Activity Assay

    Structure of p53 proteins in different neuroblastoma cell lines. The three functional domains are represented: TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. The wild-type p53 gene in SH-SY5Y, IMR-32 and LAN-5 cells contains 11 exons that encode 393 amino acids. In SK-N-BE(2) cells, p53 is mutated at codon 135 ( * ), which converts cysteine to phenylalanine. In IGR-N-91 cells, a duplication of exons 7-8-9 adds an additional 107 amino acids leading to a total of 500. In SK-N-AS cells, a mutation due to alternate splicing downstream of exon 9 leads to a protein of 341 amino acids whereas in IGR-NB8 cells, the p53 protein ends at 326 amino acids owing to the mutation E326STOP.
    Figure Legend Snippet: Structure of p53 proteins in different neuroblastoma cell lines. The three functional domains are represented: TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. The wild-type p53 gene in SH-SY5Y, IMR-32 and LAN-5 cells contains 11 exons that encode 393 amino acids. In SK-N-BE(2) cells, p53 is mutated at codon 135 ( * ), which converts cysteine to phenylalanine. In IGR-N-91 cells, a duplication of exons 7-8-9 adds an additional 107 amino acids leading to a total of 500. In SK-N-AS cells, a mutation due to alternate splicing downstream of exon 9 leads to a protein of 341 amino acids whereas in IGR-NB8 cells, the p53 protein ends at 326 amino acids owing to the mutation E326STOP.

    Techniques Used: Functional Assay, Binding Assay, Mutagenesis

    Western blot showing induction of p21/WAF1 protein by plasmid-recombinant expression vector of p53 variant transfected into p53-negative LAN-1 cells. LAN-1 cells were seeded onto 6-well plates. At a density of ∼60%, confluence cells were transfected with recombinant vector using Lipofectamine 2000 reagent according to the supplier's instructions (Invitrogen). To ascertain the transfection efficiency, cells were transfected in parallel experiments with pEGFP-C1 vector (Promega). The empty vector was used as a control. As shown in Figure 1 , note that the p53 protein from the IGR-N-91 cells analyzed by SDS–PAGE migrated more slowly than the wild-type p53 due to duplication of exons 7-8-9 as described previously ( 13 ).
    Figure Legend Snippet: Western blot showing induction of p21/WAF1 protein by plasmid-recombinant expression vector of p53 variant transfected into p53-negative LAN-1 cells. LAN-1 cells were seeded onto 6-well plates. At a density of ∼60%, confluence cells were transfected with recombinant vector using Lipofectamine 2000 reagent according to the supplier's instructions (Invitrogen). To ascertain the transfection efficiency, cells were transfected in parallel experiments with pEGFP-C1 vector (Promega). The empty vector was used as a control. As shown in Figure 1 , note that the p53 protein from the IGR-N-91 cells analyzed by SDS–PAGE migrated more slowly than the wild-type p53 due to duplication of exons 7-8-9 as described previously ( 13 ).

    Techniques Used: Western Blot, Plasmid Preparation, Recombinant, Expressing, Variant Assay, Transfection, SDS Page

    14) Product Images from "Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization"

    Article Title: Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization

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

    doi: 10.1073/pnas.0711044105

    Viability of ED43/JFH1 recombinants with putative adaptive mutations. ( A and B ) Transfection of RNA transcripts from pJ6/JFH, pED43/JFH1-GND, and pED43/JFH1-β and -γ constructs with or without mutations observed in first passage. ( A ) Percentage of Core-positive cells. Day 5 supernatants of relevant cultures were used for inoculation of first viral passages (arrow). ( B ) TCID 50 determinations on transfection supernatants. ( C ) Transfection of RNA transcripts from pJ6/JFH, pED43/JFH1-GND, and pED43/JFH1-γ T977S as well as pED43/JFH1-β T827A and pED43/JFH1-γ T827A,T977S constructs with or without mutations observed in third passage. TCID 50 determinations on transfection supernatants are shown. †, none (TCID 50 = 0); *, one (TCID 50
    Figure Legend Snippet: Viability of ED43/JFH1 recombinants with putative adaptive mutations. ( A and B ) Transfection of RNA transcripts from pJ6/JFH, pED43/JFH1-GND, and pED43/JFH1-β and -γ constructs with or without mutations observed in first passage. ( A ) Percentage of Core-positive cells. Day 5 supernatants of relevant cultures were used for inoculation of first viral passages (arrow). ( B ) TCID 50 determinations on transfection supernatants. ( C ) Transfection of RNA transcripts from pJ6/JFH, pED43/JFH1-GND, and pED43/JFH1-γ T977S as well as pED43/JFH1-β T827A and pED43/JFH1-γ T827A,T977S constructs with or without mutations observed in third passage. TCID 50 determinations on transfection supernatants are shown. †, none (TCID 50 = 0); *, one (TCID 50

    Techniques Used: Transfection, Construct

    Blocking of CD81 inhibits ED43/JFH1 infection. Huh7.5 cells were preincubated with anti-CD81 antibody or anti-HIV-p24 isotype-matched control antibody before addition of ≈100 TCID 50 ED43/JFH1-γ third-passage virus. The count of FFUs per well after an incubation period of 2 days is indicated. Each data point represents triplicate experiments. Error bars indicate standard errors of the mean. nd, not determined.
    Figure Legend Snippet: Blocking of CD81 inhibits ED43/JFH1 infection. Huh7.5 cells were preincubated with anti-CD81 antibody or anti-HIV-p24 isotype-matched control antibody before addition of ≈100 TCID 50 ED43/JFH1-γ third-passage virus. The count of FFUs per well after an incubation period of 2 days is indicated. Each data point represents triplicate experiments. Error bars indicate standard errors of the mean. nd, not determined.

    Techniques Used: Blocking Assay, Infection, Incubation

    15) Product Images from "Heme Oxygenase Isoforms Differ in Their Subcellular Trafficking during Hypoxia and Are Differentially Modulated by Cytochrome P450 Reductase"

    Article Title: Heme Oxygenase Isoforms Differ in Their Subcellular Trafficking during Hypoxia and Are Differentially Modulated by Cytochrome P450 Reductase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035483

    FLIM measurements of CFP-HOs with CPR-YFP in HEK293 cells. Pictures of the HO-CPR co-transfections were made in the CFP channel (left panels) and YFP channel (middle panels). The right panels show the color coded lifetime of the donor CFP. The corresponding color scale is shown in the center of the figure. The white bars correspond to 20 µm.
    Figure Legend Snippet: FLIM measurements of CFP-HOs with CPR-YFP in HEK293 cells. Pictures of the HO-CPR co-transfections were made in the CFP channel (left panels) and YFP channel (middle panels). The right panels show the color coded lifetime of the donor CFP. The corresponding color scale is shown in the center of the figure. The white bars correspond to 20 µm.

    Techniques Used: Transfection

    16) Product Images from "ASM-3 Acid Sphingomyelinase Functions as a Positive Regulator of the DAF-2/AGE-1 Signaling Pathway and Serves as a Novel Anti-Aging Target"

    Article Title: ASM-3 Acid Sphingomyelinase Functions as a Positive Regulator of the DAF-2/AGE-1 Signaling Pathway and Serves as a Novel Anti-Aging Target

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045890

    qRT-PCR for DAF-16/FOXO transcriptional activity and sod-3p::gfp reporter. (A) By qRT-PCR, mRNA expression of endogenous sod-3 gene was increased about 3-fold in the asm-3(ok1744) mutant animals as compared to the wild-type (N2) animals (T-test *P
    Figure Legend Snippet: qRT-PCR for DAF-16/FOXO transcriptional activity and sod-3p::gfp reporter. (A) By qRT-PCR, mRNA expression of endogenous sod-3 gene was increased about 3-fold in the asm-3(ok1744) mutant animals as compared to the wild-type (N2) animals (T-test *P

    Techniques Used: Quantitative RT-PCR, Activity Assay, Expressing, Mutagenesis, T-Test

    17) Product Images from "Efficient production of recombinant PP2A at a low temperature using a baculovirus expression system"

    Article Title: Efficient production of recombinant PP2A at a low temperature using a baculovirus expression system

    Journal: Biotechnology Reports

    doi: 10.1016/j.btre.2016.07.004

    The comparison of expression, purification, and phosphatase activity of recombinant protein phosphatase 2A (PP2A). (a) His ×8 -tagged PP2Acα was expressed in High Five insect cells at a temperature range of 11–31 °C for 2–6 days using a baculovirus expression system. The lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting using anti-polyhistidine peroxidase conjugate. The blots are representative of three independent experiments. The quantified graph of the blots are presented as means (n = 3) ± SD. (b) After purification, 0.2 μg of each protein was subjected to 12% SDS-PAGE and visualized using Coomassie Brilliant Blue R staining. (c) The activities of the recombinant proteins were assayed against p -NPP as substrate. All assays were performed in triplicate (unit, nmol/min). The p -value of two-tailed Student’s t -test is indicated.
    Figure Legend Snippet: The comparison of expression, purification, and phosphatase activity of recombinant protein phosphatase 2A (PP2A). (a) His ×8 -tagged PP2Acα was expressed in High Five insect cells at a temperature range of 11–31 °C for 2–6 days using a baculovirus expression system. The lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting using anti-polyhistidine peroxidase conjugate. The blots are representative of three independent experiments. The quantified graph of the blots are presented as means (n = 3) ± SD. (b) After purification, 0.2 μg of each protein was subjected to 12% SDS-PAGE and visualized using Coomassie Brilliant Blue R staining. (c) The activities of the recombinant proteins were assayed against p -NPP as substrate. All assays were performed in triplicate (unit, nmol/min). The p -value of two-tailed Student’s t -test is indicated.

    Techniques Used: Expressing, Purification, Activity Assay, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Staining, Two Tailed Test

    Purification and activity of recombinant protein phosphatase 2B (PP2B) and 2C (PP2C). (a) The recombinant catalytic subunit of human PP2B (rhPP2B) was synthesized in High Five cells by the co-infection of recombinant baculovirus encoding His ×8 -tagged hPP2B-Aα and FLAG-tagged hPP2B-Bα. The High Five cells were incubated at 27 °C for 3 days and at 19 °C for 4 days, after the infection of each recombinant baculovirus to express each recombinant protein. After purification, each protein was subjected to 13.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized using Coomassie Brilliant Blue R staining. (b) Purified rhPP2B was analyzed by Western blotting using anti-polyhistidine-peroxidase conjugate and anti-PP2B-Aα (immunogen: 25 residue, synthetic peptide corresponding to amino acids 490–514 of bovine calcineurin/PP2B Aα) for His 8x -tagged hPP2B-Aα, and anti-FLAG M2 monoclonal antibody-peroxidase conjugate and anti-PP2B-Bα for FLAG-tagged hPP2B-Bα followed by the enhanced chemiluminescence (ECL) system. (c) The rhPP2Cα was synthesized in High Five cells by infection of recombinant baculovirus encoding His 8x -tagged hPP2Cα. After purification, each protein was subjected to 12% SDS-PAGE and then visualized using Coomassie Brilliant Blue R staining. The activities of the rhPP2B (d) and rhPP2C (e) were assayed against p -NPP as a substrate. All assays were performed in triplicates (unit, nmol/min). The p -value of two-tailed Student’s t -test is indicated.
    Figure Legend Snippet: Purification and activity of recombinant protein phosphatase 2B (PP2B) and 2C (PP2C). (a) The recombinant catalytic subunit of human PP2B (rhPP2B) was synthesized in High Five cells by the co-infection of recombinant baculovirus encoding His ×8 -tagged hPP2B-Aα and FLAG-tagged hPP2B-Bα. The High Five cells were incubated at 27 °C for 3 days and at 19 °C for 4 days, after the infection of each recombinant baculovirus to express each recombinant protein. After purification, each protein was subjected to 13.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized using Coomassie Brilliant Blue R staining. (b) Purified rhPP2B was analyzed by Western blotting using anti-polyhistidine-peroxidase conjugate and anti-PP2B-Aα (immunogen: 25 residue, synthetic peptide corresponding to amino acids 490–514 of bovine calcineurin/PP2B Aα) for His 8x -tagged hPP2B-Aα, and anti-FLAG M2 monoclonal antibody-peroxidase conjugate and anti-PP2B-Bα for FLAG-tagged hPP2B-Bα followed by the enhanced chemiluminescence (ECL) system. (c) The rhPP2Cα was synthesized in High Five cells by infection of recombinant baculovirus encoding His 8x -tagged hPP2Cα. After purification, each protein was subjected to 12% SDS-PAGE and then visualized using Coomassie Brilliant Blue R staining. The activities of the rhPP2B (d) and rhPP2C (e) were assayed against p -NPP as a substrate. All assays were performed in triplicates (unit, nmol/min). The p -value of two-tailed Student’s t -test is indicated.

    Techniques Used: Purification, Activity Assay, Recombinant, Synthesized, Infection, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Western Blot, Two Tailed Test

    18) Product Images from "DNA-binding specificity of rice mariner-like transposases and interactions with Stowaway MITEs"

    Article Title: DNA-binding specificity of rice mariner-like transposases and interactions with Stowaway MITEs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki509

    Analysis and construction of OSMAR5 and OSMAR10 transposases. ( A ) Cloning of OSMAR5 and OSMAR10 transposase constructs. Genomic copies of the Osmar5 and Osmar10 transposons and an Osmar10 consensus sequence are represented. The extent of intrafamily pairwise sequence similarity (excluding gaps) is shown. Osmar5Ci lacks 763 bp of sequence downstream of the putative transposase gene due to a gap in the indica genome contig assembly. Asterisks above the Osmar10 elements represent mutations that result in stop codons within the transposases. Osmar5 and Osmar10 transposase ORFs were generated by PCR amplification of exons, removing introns in the process. OSMAR5 was assembled from exons matching those of Osmar5Bi . OSMAR10 is chimeric in origin, generated by assembly of the first exon of Osmar10B with the second exon of Osmar10A , which results in an intact ORF OSMAR10BA. ( B ) Comparison between Osmar5 and Osmar10 . TIRs are represented by solid black triangles. Transposase coding sequences are depicted as white boxes, noncoding regions as solid gray and introns as open triangles below the element. Percentages of sequence similarity are indicated for the two transposons; all other regions could not be confidently aligned.
    Figure Legend Snippet: Analysis and construction of OSMAR5 and OSMAR10 transposases. ( A ) Cloning of OSMAR5 and OSMAR10 transposase constructs. Genomic copies of the Osmar5 and Osmar10 transposons and an Osmar10 consensus sequence are represented. The extent of intrafamily pairwise sequence similarity (excluding gaps) is shown. Osmar5Ci lacks 763 bp of sequence downstream of the putative transposase gene due to a gap in the indica genome contig assembly. Asterisks above the Osmar10 elements represent mutations that result in stop codons within the transposases. Osmar5 and Osmar10 transposase ORFs were generated by PCR amplification of exons, removing introns in the process. OSMAR5 was assembled from exons matching those of Osmar5Bi . OSMAR10 is chimeric in origin, generated by assembly of the first exon of Osmar10B with the second exon of Osmar10A , which results in an intact ORF OSMAR10BA. ( B ) Comparison between Osmar5 and Osmar10 . TIRs are represented by solid black triangles. Transposase coding sequences are depicted as white boxes, noncoding regions as solid gray and introns as open triangles below the element. Percentages of sequence similarity are indicated for the two transposons; all other regions could not be confidently aligned.

    Techniques Used: Clone Assay, Construct, Sequencing, Generated, Polymerase Chain Reaction, Amplification

    19) Product Images from "MicroRNA-122 Triggers Mesenchymal-Epithelial Transition and Suppresses Hepatocellular Carcinoma Cell Motility and Invasion by Targeting RhoA"

    Article Title: MicroRNA-122 Triggers Mesenchymal-Epithelial Transition and Suppresses Hepatocellular Carcinoma Cell Motility and Invasion by Targeting RhoA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0101330

    miR-122 induced MET, and suppressed cell migration and invasion by down-regulating RhoA. ( A ) RhoA is a target gene of miR-122. The luciferase reporter assay was performed using Bel-7402 and 293T cells as described in the Materials and methods section. ( B ) miR-122 triggered MET-like cellular marker alterations through the down-regulation of RhoA. Western blot analysis comparing the expression of epithelial markers (ZO-1 and α-catenin) and mesenchymal markers (vimentin and fibronectin) in Bel-7402 cells expressing the control vector, miR-122, miR-122/RhoA-wt, and RhoA-T19N. ( C ) miR-122 inhibited cell migration and invasion in vitro through RhoA targeting. The effects of miR-122, miR-122/RhoA-wt, and RhoA-T19N on cell migration and invasion were evaluated using transwell and Matrigel-coated Boyden chambers, respectively.
    Figure Legend Snippet: miR-122 induced MET, and suppressed cell migration and invasion by down-regulating RhoA. ( A ) RhoA is a target gene of miR-122. The luciferase reporter assay was performed using Bel-7402 and 293T cells as described in the Materials and methods section. ( B ) miR-122 triggered MET-like cellular marker alterations through the down-regulation of RhoA. Western blot analysis comparing the expression of epithelial markers (ZO-1 and α-catenin) and mesenchymal markers (vimentin and fibronectin) in Bel-7402 cells expressing the control vector, miR-122, miR-122/RhoA-wt, and RhoA-T19N. ( C ) miR-122 inhibited cell migration and invasion in vitro through RhoA targeting. The effects of miR-122, miR-122/RhoA-wt, and RhoA-T19N on cell migration and invasion were evaluated using transwell and Matrigel-coated Boyden chambers, respectively.

    Techniques Used: Migration, Luciferase, Reporter Assay, Marker, Western Blot, Expressing, Plasmid Preparation, In Vitro

    20) Product Images from "FGFR3IIIS: a novel soluble FGFR3 spliced variant that modulates growth is frequently expressed in tumour cells"

    Article Title: FGFR3IIIS: a novel soluble FGFR3 spliced variant that modulates growth is frequently expressed in tumour cells

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601249

    FGFR3IIIS is rarely expressed in normal human tissue. Expression of the FGFR3IIIS was examined by RT–PCR using primer set 3 ( A ) and Southern blotting using an FGFR3IIIS-specific probe ( B ) in a number of normal human tissues. FGFR3IIIS was amplified using a primer specific to the FGFR3IIIS sequence at the exon seven out of 11 splice site, paired with a primer at the 5′ (exon 2) or 3′ (exon 17) ends of the published wild-type FGFR3IIIc sequence. Low-level expression of the FGFR3IIIS was detected in the heart (b), placenta (l) and ovary (q). The probe also bound nonspecifically (product identified in the reverse transcriptase negative control; not shown) to a smaller product in the thyroid gland (n). RT–PCR for the house-keeping gene β 2 -microglobulin ( β 2 -m) confirmed the quality of cDNA for amplification ( C ) a, brain; b, heart; c, kidney; d, spleen; e, liver; f, colon; g, lung; h, small intestine; i, Muscle; j, stomach; k, testis; l, placenta; m, salivary gland; n, thyroid gland; o, adrenal gland; p, pancreas; q, ovary; r, uterus; s, prostate; t, skin; u, peripheral blood lymphocytes; v, bone marrow; w, foetal brain; x, foetal liver: RNA from the MCF-7 cell line was included as a positive control (y).
    Figure Legend Snippet: FGFR3IIIS is rarely expressed in normal human tissue. Expression of the FGFR3IIIS was examined by RT–PCR using primer set 3 ( A ) and Southern blotting using an FGFR3IIIS-specific probe ( B ) in a number of normal human tissues. FGFR3IIIS was amplified using a primer specific to the FGFR3IIIS sequence at the exon seven out of 11 splice site, paired with a primer at the 5′ (exon 2) or 3′ (exon 17) ends of the published wild-type FGFR3IIIc sequence. Low-level expression of the FGFR3IIIS was detected in the heart (b), placenta (l) and ovary (q). The probe also bound nonspecifically (product identified in the reverse transcriptase negative control; not shown) to a smaller product in the thyroid gland (n). RT–PCR for the house-keeping gene β 2 -microglobulin ( β 2 -m) confirmed the quality of cDNA for amplification ( C ) a, brain; b, heart; c, kidney; d, spleen; e, liver; f, colon; g, lung; h, small intestine; i, Muscle; j, stomach; k, testis; l, placenta; m, salivary gland; n, thyroid gland; o, adrenal gland; p, pancreas; q, ovary; r, uterus; s, prostate; t, skin; u, peripheral blood lymphocytes; v, bone marrow; w, foetal brain; x, foetal liver: RNA from the MCF-7 cell line was included as a positive control (y).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Southern Blot, Amplification, Sequencing, Negative Control, Positive Control

    Expression of FGFR3IIIc and FGFR3IIIS in tumour cell lines and primary tumours. Expression of FGFR3IIIc (detected by RT–PCR using primer set 2) was rare in the tumour cell lines and tumours examined; however, the splice variant FGFR3IIIS was expressed in 57% (17 out of 30) of tumours (a) and 79% (15 out of 19) of the tumour cell lines (b) examined. The quality of RNA from cell lines and tumours was confirmed by amplification for the house-keeping gene β 2 -microglobulin (results not shown). ( A ) RT-PCR for FGFR3IIIS (a) rhabdomyosarcoma, (b) Wilms' tumour, (c) astrocytoma, (d and j). neuroblastoma, (e–h) Ewing's sarcoma, (i) medulloblastoma, (k) MCF7 cell line. ( B ) FGFR3IIIS RT-PCR for (a) U266, (b) IMR-32, (c) RD-ES, (d) TC-32, (e) SK-N-MC, (f) TTC-466, (g) ST118, (h) RT112, (i) LNCAP, (j) CCL136, (k) VUP, (l) RH30, (m) PC12, (n) EJ, (o) HT29, (p) MCF7 cell lines. M=molecular weight markers; RT+ve=reverse transcriptase enzyme present; RT-ve=no reverse transcriptase enzyme included in the reaction; negative control.
    Figure Legend Snippet: Expression of FGFR3IIIc and FGFR3IIIS in tumour cell lines and primary tumours. Expression of FGFR3IIIc (detected by RT–PCR using primer set 2) was rare in the tumour cell lines and tumours examined; however, the splice variant FGFR3IIIS was expressed in 57% (17 out of 30) of tumours (a) and 79% (15 out of 19) of the tumour cell lines (b) examined. The quality of RNA from cell lines and tumours was confirmed by amplification for the house-keeping gene β 2 -microglobulin (results not shown). ( A ) RT-PCR for FGFR3IIIS (a) rhabdomyosarcoma, (b) Wilms' tumour, (c) astrocytoma, (d and j). neuroblastoma, (e–h) Ewing's sarcoma, (i) medulloblastoma, (k) MCF7 cell line. ( B ) FGFR3IIIS RT-PCR for (a) U266, (b) IMR-32, (c) RD-ES, (d) TC-32, (e) SK-N-MC, (f) TTC-466, (g) ST118, (h) RT112, (i) LNCAP, (j) CCL136, (k) VUP, (l) RH30, (m) PC12, (n) EJ, (o) HT29, (p) MCF7 cell lines. M=molecular weight markers; RT+ve=reverse transcriptase enzyme present; RT-ve=no reverse transcriptase enzyme included in the reaction; negative control.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Variant Assay, Amplification, Molecular Weight, Negative Control

    Identification and characterisation of a novel FGFR3IIIc variant, FGFR3IIIS. ( A ) The FGFR3 extracellular domain, first Ig-like loop (I) and the transmembrane/intracellular domain (K) was amplified in the breast carcinoma MCF-7 (a) and Ewing's sarcoma TC-32 (b) cell lines by RT–PCR. M =φX174 RF DNA/ Hae III molecular weight marker. ( B ) Diagrammatical representation of FGFR3IIIc, FGFR3IIIS and the domains that each exon codes for. The split tyrosine-kinase domain is represented as one domain, since it is not known as to which exon codes for the region between the two domains. Sequence analysis of FGFR3IIIS demonstrated loss of exons 9 (encoding the second half of the third Ig-like domain) and 10 (coding for the transmembrane domain) and a 30 bp deletion in exon 7 (within the region between the second and third Ig-like loops, including a potential glycosylation site). Primers were designed in exons 3 and 4 to amplify the first Ig-like loop of the extracellular domain (primer set 1) and within exons 6 and 12 (primer set 2) to amplify the third Ig-like loop and the transmembrane/tyrosine kinase domain.
    Figure Legend Snippet: Identification and characterisation of a novel FGFR3IIIc variant, FGFR3IIIS. ( A ) The FGFR3 extracellular domain, first Ig-like loop (I) and the transmembrane/intracellular domain (K) was amplified in the breast carcinoma MCF-7 (a) and Ewing's sarcoma TC-32 (b) cell lines by RT–PCR. M =φX174 RF DNA/ Hae III molecular weight marker. ( B ) Diagrammatical representation of FGFR3IIIc, FGFR3IIIS and the domains that each exon codes for. The split tyrosine-kinase domain is represented as one domain, since it is not known as to which exon codes for the region between the two domains. Sequence analysis of FGFR3IIIS demonstrated loss of exons 9 (encoding the second half of the third Ig-like domain) and 10 (coding for the transmembrane domain) and a 30 bp deletion in exon 7 (within the region between the second and third Ig-like loops, including a potential glycosylation site). Primers were designed in exons 3 and 4 to amplify the first Ig-like loop of the extracellular domain (primer set 1) and within exons 6 and 12 (primer set 2) to amplify the third Ig-like loop and the transmembrane/tyrosine kinase domain.

    Techniques Used: Variant Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker, Sequencing

    21) Product Images from "Unspliced Precursors of NMD-Sensitive ?-Globin Transcripts Exhibit Decreased Steady-State Levels in Erythroid Cells"

    Article Title: Unspliced Precursors of NMD-Sensitive ?-Globin Transcripts Exhibit Decreased Steady-State Levels in Erythroid Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038505

    The nonsense codon effect on the β-globin pre-mRNA abundance exhibits cell line specificity. (A) HeLa cells were stably transfected with the βWT or β39 constructs as indicated below the histogram. Total RNA was isolated and βWT and β39 steady-state mRNA levels were quantified by RT-qPCR using specific primers for the human β-globin processed mRNA, as in Figure 5C, D . The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (B) Total RNA was also analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as in Figure 5A, B . For each case, intron 1 and intron 2 containing human β-globin pre-RNA levels were determined by normalization to the level of the puromycin resistance mRNA, using the comparative Ct method, and compared to the wild-type control. The percentage pre-mRNA values were plotted for each construct and the histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in ( A ).
    Figure Legend Snippet: The nonsense codon effect on the β-globin pre-mRNA abundance exhibits cell line specificity. (A) HeLa cells were stably transfected with the βWT or β39 constructs as indicated below the histogram. Total RNA was isolated and βWT and β39 steady-state mRNA levels were quantified by RT-qPCR using specific primers for the human β-globin processed mRNA, as in Figure 5C, D . The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (B) Total RNA was also analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as in Figure 5A, B . For each case, intron 1 and intron 2 containing human β-globin pre-RNA levels were determined by normalization to the level of the puromycin resistance mRNA, using the comparative Ct method, and compared to the wild-type control. The percentage pre-mRNA values were plotted for each construct and the histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in ( A ).

    Techniques Used: Stable Transfection, Transfection, Construct, Isolation, Quantitative RT-PCR, Two Tailed Test, Real-time Polymerase Chain Reaction

    The decreased β-globin pre-mRNA levels are specific for transcripts carrying NMD-competent nonsense mutations. (A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrows represent the position of the nonsense mutations at codon 26 (GAG→UAG; β26), 39 (CAG→UAG; β39), 62 (GCT→UAG; β62) or 127 (CAG→UAG; β127). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m 7 G) and poly(A) tail [(A) n ] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods ). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in ( A ). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods ). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in ( B ). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in ( C ). Statistical analysis was performed using Student's t test (unpaired, two-tailed).
    Figure Legend Snippet: The decreased β-globin pre-mRNA levels are specific for transcripts carrying NMD-competent nonsense mutations. (A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrows represent the position of the nonsense mutations at codon 26 (GAG→UAG; β26), 39 (CAG→UAG; β39), 62 (GCT→UAG; β62) or 127 (CAG→UAG; β127). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m 7 G) and poly(A) tail [(A) n ] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods ). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in ( A ). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods ). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in ( B ). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in ( C ). Statistical analysis was performed using Student's t test (unpaired, two-tailed).

    Techniques Used: Stable Transfection, Recombinase Polymerase Amplification, Transfection, Construct, Isolation, Two Tailed Test

    The low levels of the β39 pre-mRNA are not due to the disruption of a regulatory element encompassing codon 39. (A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrow represents the position of the nonsense (CAG→UAG) or missense (CAG→GAG) mutation at codon 39 (β39 and β39missense respectively). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m 7 G) and poly(A) tail [(A) n ] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods ). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in ( A ). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods ). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in ( B ). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in (C) .
    Figure Legend Snippet: The low levels of the β39 pre-mRNA are not due to the disruption of a regulatory element encompassing codon 39. (A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrow represents the position of the nonsense (CAG→UAG) or missense (CAG→GAG) mutation at codon 39 (β39 and β39missense respectively). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m 7 G) and poly(A) tail [(A) n ] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods ). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in ( A ). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods ). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in ( B ). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in (C) .

    Techniques Used: Stable Transfection, Mutagenesis, Recombinase Polymerase Amplification, Transfection, Construct, Isolation, Two Tailed Test

    The presence of the nonsense codon equally decreases the abundance of intron 1 versus intron 2 containing human β-globin pre-mRNAs. (A) Schematic representation of the human β-globin pre-mRNA, as in Figures 3D and 4D . The two pairs of arrows represent the coordinates of both amplicons obtained in the qPCR reactions: intron1-exon2 and exon2-intron2 amplicons. (B) MEL cells were stably transfected with a test human β-globin construct as specified below the histogram. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as shown in ( A ). For each case, intron 1 and intron 2 containing human β-globin pre-RNAs levels were determined by normalization to the level of murine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, using the comparative C t method, and compared to the wild-type control. The percentage pre-mRNA values were plotted for each construct and the histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (C) Schematic representation of the studied human β-globin mRNAs as in Figures 3A and 4A . The pair of arrows represents the coordinates of the amplicon obtained in the qPCR reactions: exon2–3 amplicon. (D) Human β-globin mRNA quantification was performed by RT-qPCR as in ( B ), but using specific primers for the human β-globin processed mRNA. Levels of each human β-globin mRNA variant were determined by normalization to the level of murine GAPDH mRNA, using the comparative Ct method, and compared to the wild-type control. The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in ( B ).
    Figure Legend Snippet: The presence of the nonsense codon equally decreases the abundance of intron 1 versus intron 2 containing human β-globin pre-mRNAs. (A) Schematic representation of the human β-globin pre-mRNA, as in Figures 3D and 4D . The two pairs of arrows represent the coordinates of both amplicons obtained in the qPCR reactions: intron1-exon2 and exon2-intron2 amplicons. (B) MEL cells were stably transfected with a test human β-globin construct as specified below the histogram. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as shown in ( A ). For each case, intron 1 and intron 2 containing human β-globin pre-RNAs levels were determined by normalization to the level of murine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, using the comparative C t method, and compared to the wild-type control. The percentage pre-mRNA values were plotted for each construct and the histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (C) Schematic representation of the studied human β-globin mRNAs as in Figures 3A and 4A . The pair of arrows represents the coordinates of the amplicon obtained in the qPCR reactions: exon2–3 amplicon. (D) Human β-globin mRNA quantification was performed by RT-qPCR as in ( B ), but using specific primers for the human β-globin processed mRNA. Levels of each human β-globin mRNA variant were determined by normalization to the level of murine GAPDH mRNA, using the comparative Ct method, and compared to the wild-type control. The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in ( B ).

    Techniques Used: Real-time Polymerase Chain Reaction, Stable Transfection, Transfection, Construct, Isolation, Quantitative RT-PCR, Two Tailed Test, Amplification, Variant Assay

    22) Product Images from "Expression of C-terminal deleted p53 isoforms in neuroblastoma"

    Article Title: Expression of C-terminal deleted p53 isoforms in neuroblastoma

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl619

    Detection of p53 mRNA abnormalities in SK-N-AS (AS) in comparison with SH-SY5Y (SH) cells. ( A ) Amplification of p53 cDNA using primers from exons 8 to 10 as indicated below each arrow; for precise position see Table 1 and GenBank K03199: F2 (forward primer in exon 8); R3 (reverse primer at the junction of exon 8/9); R4 (the first moiety of exon 9); R5 (exon 9, 30 nt downstream R4); R6 (beginning of exon 10). Note that no amplification was observed in SK-N-AS from exon 10 (last lane), in contrast to SH-SY5Y. ( B ) RT–PCR from SK-N-AS compared to SH-SY5Y cells. Specific primers ( Table 1 ) were used to amplify the DBD, the p53β isoform (β) and the C-terminal domain (C-ter).
    Figure Legend Snippet: Detection of p53 mRNA abnormalities in SK-N-AS (AS) in comparison with SH-SY5Y (SH) cells. ( A ) Amplification of p53 cDNA using primers from exons 8 to 10 as indicated below each arrow; for precise position see Table 1 and GenBank K03199: F2 (forward primer in exon 8); R3 (reverse primer at the junction of exon 8/9); R4 (the first moiety of exon 9); R5 (exon 9, 30 nt downstream R4); R6 (beginning of exon 10). Note that no amplification was observed in SK-N-AS from exon 10 (last lane), in contrast to SH-SY5Y. ( B ) RT–PCR from SK-N-AS compared to SH-SY5Y cells. Specific primers ( Table 1 ) were used to amplify the DBD, the p53β isoform (β) and the C-terminal domain (C-ter).

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction

    23) Product Images from "Activation-induced deaminase heterozygous MRL/lpr mice are delayed in the production of high-affinity pathogenic antibodies and in the development of lupus nephritis"

    Article Title: Activation-induced deaminase heterozygous MRL/lpr mice are delayed in the production of high-affinity pathogenic antibodies and in the development of lupus nephritis

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2008.02882.x

    Reduced somatic hypermutation in activation-induced deaminase (AID) heterozygotes in the absence of selection. Peyer's patches were collected from AID wild-type (WT) and heterozygous (Het) MRL/lpr mice at 8, 12 and 16–18 weeks of age ( n = 6, n = 6 and n = 18, respectively). Peyer's patches from mice with the same genotypes and ages were pooled for the 8- and 12-week-old age groups but not the 16- to 18-week-old mice, in which mice were analysed individually. B220 + CD19 + GL7 + cells were sorted and lysed for DNA preparation. A 1·2-kb fragment from the intronic region 3′ of the rearranged endogenous V H genes was amplified by polymerase chain reaction and cloned for DNA sequencing. Totals of 46 (8 weeks), 40 (12 weeks), and 179 (16–18 weeks) sequences were analysed with approximately the same number of clones for heterozygotes and wild-type mice. The mutation frequency was calculated among all clones (mutations per clone) and among only those clones that were mutated at least once (mutations per mutated clone). Error bars represents standard errors.
    Figure Legend Snippet: Reduced somatic hypermutation in activation-induced deaminase (AID) heterozygotes in the absence of selection. Peyer's patches were collected from AID wild-type (WT) and heterozygous (Het) MRL/lpr mice at 8, 12 and 16–18 weeks of age ( n = 6, n = 6 and n = 18, respectively). Peyer's patches from mice with the same genotypes and ages were pooled for the 8- and 12-week-old age groups but not the 16- to 18-week-old mice, in which mice were analysed individually. B220 + CD19 + GL7 + cells were sorted and lysed for DNA preparation. A 1·2-kb fragment from the intronic region 3′ of the rearranged endogenous V H genes was amplified by polymerase chain reaction and cloned for DNA sequencing. Totals of 46 (8 weeks), 40 (12 weeks), and 179 (16–18 weeks) sequences were analysed with approximately the same number of clones for heterozygotes and wild-type mice. The mutation frequency was calculated among all clones (mutations per clone) and among only those clones that were mutated at least once (mutations per mutated clone). Error bars represents standard errors.

    Techniques Used: Activation Assay, Selection, Mouse Assay, Amplification, Polymerase Chain Reaction, Clone Assay, DNA Sequencing, Mutagenesis

    24) Product Images from "A genome scan for milk production traits in dairy goats reveals two new mutations in Dgat1 reducing milk fat content"

    Article Title: A genome scan for milk production traits in dairy goats reveals two new mutations in Dgat1 reducing milk fat content

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-02052-0

    Determination of DGAT1 gene structure and polymorphism. The intron/exon structure of the LT221856 sequence is shown together with the SNPs detected and the primers used for sequencing. The location of the remaining N stretches is shown, together with their length (bp). A zoom on the coding region is also shown. The red arrows indicate the position of the R251L and R396W mutations.
    Figure Legend Snippet: Determination of DGAT1 gene structure and polymorphism. The intron/exon structure of the LT221856 sequence is shown together with the SNPs detected and the primers used for sequencing. The location of the remaining N stretches is shown, together with their length (bp). A zoom on the coding region is also shown. The red arrows indicate the position of the R251L and R396W mutations.

    Techniques Used: Sequencing

    Effect of R396W genotype of DGAT1 gene on fat content (FC), quantity of saturated fatty acids (SFA) in milk, quantity of unsaturated fatty acids (UFA) in milk and fat yield (FY). The LS means have been estimated by using a mixed model including the genotype and the sire effect. Error bars indicate standard errors. Traits are expressed as the standard deviation of yield deviations. Lower case letters show significant differences in the trait between genotypes, as determined by a t-test at p
    Figure Legend Snippet: Effect of R396W genotype of DGAT1 gene on fat content (FC), quantity of saturated fatty acids (SFA) in milk, quantity of unsaturated fatty acids (UFA) in milk and fat yield (FY). The LS means have been estimated by using a mixed model including the genotype and the sire effect. Error bars indicate standard errors. Traits are expressed as the standard deviation of yield deviations. Lower case letters show significant differences in the trait between genotypes, as determined by a t-test at p

    Techniques Used: Standard Deviation

    Quantity of triglyceride produced in each combination of reaction time and enzyme quantity for the four DGAT1 constructs: wild type, with the R251L mutation, with the R396W mutation, and with both mutations. This quantity of triglyceride has been corrected by an internal standard (TG19) and by the amount of measured cholesterol. The small symbols correspond to individual measurements and the large symbols are the mean of the corresponding group.
    Figure Legend Snippet: Quantity of triglyceride produced in each combination of reaction time and enzyme quantity for the four DGAT1 constructs: wild type, with the R251L mutation, with the R396W mutation, and with both mutations. This quantity of triglyceride has been corrected by an internal standard (TG19) and by the amount of measured cholesterol. The small symbols correspond to individual measurements and the large symbols are the mean of the corresponding group.

    Techniques Used: Produced, Construct, Mutagenesis

    25) Product Images from "A Targeting Modality for Destruction of RNA Polymerase I that Possesses Anticancer Activity"

    Article Title: A Targeting Modality for Destruction of RNA Polymerase I that Possesses Anticancer Activity

    Journal: Cancer cell

    doi: 10.1016/j.ccr.2013.12.009

    BMH-21 Causes Destruction of RPA194
    Figure Legend Snippet: BMH-21 Causes Destruction of RPA194

    Techniques Used:

    Inhibition of RPA194 Degradation does not Rescue Transcription
    Figure Legend Snippet: Inhibition of RPA194 Degradation does not Rescue Transcription

    Techniques Used: Inhibition

    Loss of RPA194 Correlates with Decreased Cancer Cell Viability
    Figure Legend Snippet: Loss of RPA194 Correlates with Decreased Cancer Cell Viability

    Techniques Used:

    26) Product Images from "ASM-3 Acid Sphingomyelinase Functions as a Positive Regulator of the DAF-2/AGE-1 Signaling Pathway and Serves as a Novel Anti-Aging Target"

    Article Title: ASM-3 Acid Sphingomyelinase Functions as a Positive Regulator of the DAF-2/AGE-1 Signaling Pathway and Serves as a Novel Anti-Aging Target

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045890

    Loss of asm induces the nuclear localization of DAF-16::GFP fusion protein and affects the DAF-16 protein levels. (A) and (B) DAF-16::GFP cellular distributions were examined by fluorescence microscopy and tail regions of animals were shown here. For images on the body and head regions of the animals, see Figure S5 . Animals were examined on adult day 1 (A) or day 4 (B). (A) In the rrf-3(pk1426);daf-16::gfp mutant background, vector control (L4440) RNAi showed that DAF-16::GFP diffusely localized in the cytoplasm, while asm-3 , asm-1 or asm-2 RNAi each induced the nuclear localization of DAF-16::GFP. RNAi inactivation of daf-2 and age-1 (positive controls) and RNAi inactivation of daf-16 and daf-18 (negative controls) were carried out in parallel. (B) In the asm-3(ok1744);rrf- 3(pk1426);daf-16::gfp mutant background, RNAi knockdown of asm-1 , asm-2 or asm-1/asm-2 (double RNAi of asm-1 and asm-2 ) further induced the nuclear localization of DAF-16::GFP protein. (C) and (D) western blot analysis of endogenous DAF-16 protein levels. (C) Increased DAF-16 protein levels were observed in the asm-3(ok1744) and daf-2(e1370) mutants as compared with that of wild-type control. Lysates were prepared from adult day 1 animals. (D) RNAi knockdown of asm-3 or daf-2 each elevated DAF-16 protein level as compared with that of vector control (L4440) RNAi. The specificity of the immunodetection was verified by the disappearance of DAF-16 protein in the daf-16(mgDf47) null mutants or in animals treated with daf-16 RNAi. Lysates were prepared from RNAi-treated, adult day 2 animals. In (C) and (D), quantification of the relative abundance of DAF-16 proteins was shown with the DAF-16 protein levels being normalized against the beta-actin protein levels using the ImageJ software.
    Figure Legend Snippet: Loss of asm induces the nuclear localization of DAF-16::GFP fusion protein and affects the DAF-16 protein levels. (A) and (B) DAF-16::GFP cellular distributions were examined by fluorescence microscopy and tail regions of animals were shown here. For images on the body and head regions of the animals, see Figure S5 . Animals were examined on adult day 1 (A) or day 4 (B). (A) In the rrf-3(pk1426);daf-16::gfp mutant background, vector control (L4440) RNAi showed that DAF-16::GFP diffusely localized in the cytoplasm, while asm-3 , asm-1 or asm-2 RNAi each induced the nuclear localization of DAF-16::GFP. RNAi inactivation of daf-2 and age-1 (positive controls) and RNAi inactivation of daf-16 and daf-18 (negative controls) were carried out in parallel. (B) In the asm-3(ok1744);rrf- 3(pk1426);daf-16::gfp mutant background, RNAi knockdown of asm-1 , asm-2 or asm-1/asm-2 (double RNAi of asm-1 and asm-2 ) further induced the nuclear localization of DAF-16::GFP protein. (C) and (D) western blot analysis of endogenous DAF-16 protein levels. (C) Increased DAF-16 protein levels were observed in the asm-3(ok1744) and daf-2(e1370) mutants as compared with that of wild-type control. Lysates were prepared from adult day 1 animals. (D) RNAi knockdown of asm-3 or daf-2 each elevated DAF-16 protein level as compared with that of vector control (L4440) RNAi. The specificity of the immunodetection was verified by the disappearance of DAF-16 protein in the daf-16(mgDf47) null mutants or in animals treated with daf-16 RNAi. Lysates were prepared from RNAi-treated, adult day 2 animals. In (C) and (D), quantification of the relative abundance of DAF-16 proteins was shown with the DAF-16 protein levels being normalized against the beta-actin protein levels using the ImageJ software.

    Techniques Used: Fluorescence, Microscopy, Mutagenesis, Plasmid Preparation, Western Blot, Immunodetection, Software

    27) Product Images from "Functional analysis of Flavonoid 3′,5′-hydroxylase from Tea plant (Camellia sinensis): critical role in the accumulation of catechins"

    Article Title: Functional analysis of Flavonoid 3′,5′-hydroxylase from Tea plant (Camellia sinensis): critical role in the accumulation of catechins

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-014-0347-7

    Expression of CsF3′5′H in different tea tissues. (A) Relative expression of CsF3′5′H in different tea tissues analyzed by qRT-PCR and Semi-quantitative RT PCR for Cs F3′5′H and GAPDH in different tea tissues. (B) Relative expression of CsF3′5′H in different light and sucrose conditions analyzed by qRT-PCR and Semi-quantitative RT PCR for GAPDH and Cs F3′5′H in different light and sucrose conditions. The data represent the mean ± SD from three independent measurements. The different letters (a, b, c, d) and *indicated the significant level at P
    Figure Legend Snippet: Expression of CsF3′5′H in different tea tissues. (A) Relative expression of CsF3′5′H in different tea tissues analyzed by qRT-PCR and Semi-quantitative RT PCR for Cs F3′5′H and GAPDH in different tea tissues. (B) Relative expression of CsF3′5′H in different light and sucrose conditions analyzed by qRT-PCR and Semi-quantitative RT PCR for GAPDH and Cs F3′5′H in different light and sucrose conditions. The data represent the mean ± SD from three independent measurements. The different letters (a, b, c, d) and *indicated the significant level at P

    Techniques Used: Expressing, Quantitative RT-PCR

    Flower color after overexpression of CsF3′5′H and qRT-PCR of transgenic tobacco plants. (A) Tobacco flowers of wild-type (CK) and CsF3′5′H transgenes (Line 1). (B) Tobacco flowers of wild-type (CK), CsF3′5′H transgenes (Line 1, 3, 9 and 15)and qRT-PCR for CsF3′5′H in flowers from CK and transgenic lines. (C) HPLC chromatograms of anthocyanidins (at 530 nm) and flavonol glacosides (at 340 nm) in tobacco flowers from CK and Line 1 (1: DEL; 2: CYA; 3: quercetin-3-O-rutinoside, 4: kaempferol-3-O-rutinoside). (D) Concentration of anthocyanidins in tobacco flowers from CK, CsF3′5′H transgenes (Line 1, 3 9, and 15) and vector control. The data represent the mean ± SD from three independent measurements. (E) qRT-PCR for flavonoid-related genes in tobacco flowers from CK, CsF3′5′H transgenes (Line 1, Line3 Line 9) and vector control. *indicated the significant level at P
    Figure Legend Snippet: Flower color after overexpression of CsF3′5′H and qRT-PCR of transgenic tobacco plants. (A) Tobacco flowers of wild-type (CK) and CsF3′5′H transgenes (Line 1). (B) Tobacco flowers of wild-type (CK), CsF3′5′H transgenes (Line 1, 3, 9 and 15)and qRT-PCR for CsF3′5′H in flowers from CK and transgenic lines. (C) HPLC chromatograms of anthocyanidins (at 530 nm) and flavonol glacosides (at 340 nm) in tobacco flowers from CK and Line 1 (1: DEL; 2: CYA; 3: quercetin-3-O-rutinoside, 4: kaempferol-3-O-rutinoside). (D) Concentration of anthocyanidins in tobacco flowers from CK, CsF3′5′H transgenes (Line 1, 3 9, and 15) and vector control. The data represent the mean ± SD from three independent measurements. (E) qRT-PCR for flavonoid-related genes in tobacco flowers from CK, CsF3′5′H transgenes (Line 1, Line3 Line 9) and vector control. *indicated the significant level at P

    Techniques Used: Over Expression, Quantitative RT-PCR, Transgenic Assay, High Performance Liquid Chromatography, Concentration Assay, Plasmid Preparation

    28) Product Images from "NSAID-activated gene-1 as a molecular target for capsaicin-induced apoptosis through a novel molecular mechanism involving GSK3?, C/EBP? and ATF3"

    Article Title: NSAID-activated gene-1 as a molecular target for capsaicin-induced apoptosis through a novel molecular mechanism involving GSK3?, C/EBP? and ATF3

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgq016

    GSK3β and PKCδ mediate C/EBPβ phosphorylation and capsaicin-induced NAG-1 expression. (A) HCT-116 cells were pretreated with different tyrosine kinases (RO-31-8220, 2.5 μM; PD98059, 40 μM; SP600125, 30 μM; SB203580, 15 μM; AG490, 50 μM; MG132, 10 μM; wortmannin, 0.5 μM; AR-A014418, 30 μM; rapamycin, 0.1 μM) for 30 min and treated with 50 μM capsaicin for 7 h. Western blot was performed for NAG-1 and actin. (B) HCT-116 cells were pretreated with RO-31-8220 (2.5 μM) and rottlerin (10 μM) for 30 min and treated with 50 μM capsaicin for 7 h; then western blot was performed for NAG-1 and actin. (C) HCT-116 cells were transfected with control or siRNA of GSK3β for 24 h and then treated with 50 μM of capsaicin for 24 h; western blot was performed for GSK3β, NAG-1 and actin. (D) HCT-116 cells were treated with 50 μM capsaicin for 7 h, and the cell lysate was pulled down with C/EBPβ antibody. Western blot was performed for phosphor Ser/Thr and C/EBPβ as described in Materials and Methods. (E) HCT-116 cells were pretreated with RO-31-8220 (2.5 μM) or AR-A014418 (30 μM) for 30 min and then treated with 50 μM capsaicin for 2 h or 7 h. The cell lysate was immunoprecipiated using C/EBPβ antibody, and western blot was performed for phosphor Ser/Thr and C/EBPβ as described in Materials and Methods.
    Figure Legend Snippet: GSK3β and PKCδ mediate C/EBPβ phosphorylation and capsaicin-induced NAG-1 expression. (A) HCT-116 cells were pretreated with different tyrosine kinases (RO-31-8220, 2.5 μM; PD98059, 40 μM; SP600125, 30 μM; SB203580, 15 μM; AG490, 50 μM; MG132, 10 μM; wortmannin, 0.5 μM; AR-A014418, 30 μM; rapamycin, 0.1 μM) for 30 min and treated with 50 μM capsaicin for 7 h. Western blot was performed for NAG-1 and actin. (B) HCT-116 cells were pretreated with RO-31-8220 (2.5 μM) and rottlerin (10 μM) for 30 min and treated with 50 μM capsaicin for 7 h; then western blot was performed for NAG-1 and actin. (C) HCT-116 cells were transfected with control or siRNA of GSK3β for 24 h and then treated with 50 μM of capsaicin for 24 h; western blot was performed for GSK3β, NAG-1 and actin. (D) HCT-116 cells were treated with 50 μM capsaicin for 7 h, and the cell lysate was pulled down with C/EBPβ antibody. Western blot was performed for phosphor Ser/Thr and C/EBPβ as described in Materials and Methods. (E) HCT-116 cells were pretreated with RO-31-8220 (2.5 μM) or AR-A014418 (30 μM) for 30 min and then treated with 50 μM capsaicin for 2 h or 7 h. The cell lysate was immunoprecipiated using C/EBPβ antibody, and western blot was performed for phosphor Ser/Thr and C/EBPβ as described in Materials and Methods.

    Techniques Used: Expressing, Western Blot, Transfection

    DNA binding activity of C/EBPβ in the NAG-1 promoter. (A) HCT-116 cells were treated with ethanol (E) or 50 μM of capsaicin (Cap) for 6 h, and nuclear extracts were prepared. Biotin-labeled oligonucleotide (100 nM) was incubated with nuclear protein (5 μg) and 1× binding buffer (Promega) at room temperature for 20 min, as described in Materials and Methods. Oligonucleotide probes contained the following sequences: NAG-1-110/-103: 5′-gctgtggtcattgctgtggtcattgctgtggtcatt-3′ and NAG-1-87/-80: 5′- tctgcaggcaggtctgcaggcaggtctgcaggcagg-3′. The specific DNA–protein complexes are indicated by arrows. NE, nuclear extract. (B) The competition of the DNA binding was obtained using a 10 and 100-time excess of the unlabeled oligonucleotide (lane 3–4, 7–8). Nuclear protein (5 μg) was preincubated with the unlabeled oligonucleotide (10- or 100-fold) for 10 min and then incubated with biotin-labeled oligonucleotide and 1× binding buffer (Promega) at room temperature for 20 min. (C) The in vitro synthesized C/EBPβ protein was used for competition study as described in (B). For C/EBPβ synthesis, in vitro translation was performed with pcDNA3.1/C/EBPβ2/V5/His using TNT Quick Coupled Transcription/Translation Systems (Promega). IVT, in vitro translated protein.
    Figure Legend Snippet: DNA binding activity of C/EBPβ in the NAG-1 promoter. (A) HCT-116 cells were treated with ethanol (E) or 50 μM of capsaicin (Cap) for 6 h, and nuclear extracts were prepared. Biotin-labeled oligonucleotide (100 nM) was incubated with nuclear protein (5 μg) and 1× binding buffer (Promega) at room temperature for 20 min, as described in Materials and Methods. Oligonucleotide probes contained the following sequences: NAG-1-110/-103: 5′-gctgtggtcattgctgtggtcattgctgtggtcatt-3′ and NAG-1-87/-80: 5′- tctgcaggcaggtctgcaggcaggtctgcaggcagg-3′. The specific DNA–protein complexes are indicated by arrows. NE, nuclear extract. (B) The competition of the DNA binding was obtained using a 10 and 100-time excess of the unlabeled oligonucleotide (lane 3–4, 7–8). Nuclear protein (5 μg) was preincubated with the unlabeled oligonucleotide (10- or 100-fold) for 10 min and then incubated with biotin-labeled oligonucleotide and 1× binding buffer (Promega) at room temperature for 20 min. (C) The in vitro synthesized C/EBPβ protein was used for competition study as described in (B). For C/EBPβ synthesis, in vitro translation was performed with pcDNA3.1/C/EBPβ2/V5/His using TNT Quick Coupled Transcription/Translation Systems (Promega). IVT, in vitro translated protein.

    Techniques Used: Binding Assay, Activity Assay, Labeling, Incubation, In Vitro, Synthesized

    Capsaicin enhances GSK3β expression and protein interactions of GSK3β, C/EBPβ, and ATF3. (A) HCT-116 cells were treated with indicated concentrations of capsaicin for 24 or 48 h and western blot was performed for GSK3β and actin. (B) HCT-116 cells were treated with 50 μM of capsaicin for indicated time points, and western blot was performed for GSK3β and actin. (C) The cells were treated with 50 μM of capsaicin for 6 h and nuclear and cytosol fractions were isolated, and then western blot was performed for GSK3β, C/EBPβ, ATF3, and actin. (D) The cells were transfected with C/EBPβ expression vector-tagged V5 (pcDNA3.1/C/EBPβ2/V5/His) and treated with 50 μM capsaicin for 6 h. The cell lysates were immunoprecipitated with ProBond nickel-chelating resin (Invitrogen), separated by sodium dodecyl sulfate–polyacrylamide gel, transferred to membranes, and then immunoblotted with V5, GSK3β or ATF3 antibody. (E) HCT-116 cells were transfected with pcDNA3.1/C/EBPβ2/V5/His, pretreated with RO-31-8220 (2.5 μM) or AR-A014418 (30 μM) for 30 min and then treated with 50 μM capsaicin for 2 or 6 h. Immunoprecipitation was performed as described in (D). (F) The cells were transfected with GSK3β expression vector (pcDNA3.1/GSK3β/V5/His) and treated with 50 μM capsaicin for 6 h. The cell lysates were immunoprecipitated with ProBond nickel-chelating resin (Invitrogen), separated by sodium dodecyl sulfate–polyacrylamide gel, transferred to membranes, and then immunoblotted with V5, C/EBPβ or ATF3 antibody. (G) Chromatin immunoprecipitation assay for ATF3 binding was performed using a DNA–protein complex treated with 50 μM capsaicin for 24 h as described in Materials and Methods. The sequence of the NAG-1 promoter region (−131/+137) was amplified by PCR primer pairs as indicated by the arrows. The input represents PCR products obtained from 1% aliquots of chromatin pellets escaping immunoprecipitation.
    Figure Legend Snippet: Capsaicin enhances GSK3β expression and protein interactions of GSK3β, C/EBPβ, and ATF3. (A) HCT-116 cells were treated with indicated concentrations of capsaicin for 24 or 48 h and western blot was performed for GSK3β and actin. (B) HCT-116 cells were treated with 50 μM of capsaicin for indicated time points, and western blot was performed for GSK3β and actin. (C) The cells were treated with 50 μM of capsaicin for 6 h and nuclear and cytosol fractions were isolated, and then western blot was performed for GSK3β, C/EBPβ, ATF3, and actin. (D) The cells were transfected with C/EBPβ expression vector-tagged V5 (pcDNA3.1/C/EBPβ2/V5/His) and treated with 50 μM capsaicin for 6 h. The cell lysates were immunoprecipitated with ProBond nickel-chelating resin (Invitrogen), separated by sodium dodecyl sulfate–polyacrylamide gel, transferred to membranes, and then immunoblotted with V5, GSK3β or ATF3 antibody. (E) HCT-116 cells were transfected with pcDNA3.1/C/EBPβ2/V5/His, pretreated with RO-31-8220 (2.5 μM) or AR-A014418 (30 μM) for 30 min and then treated with 50 μM capsaicin for 2 or 6 h. Immunoprecipitation was performed as described in (D). (F) The cells were transfected with GSK3β expression vector (pcDNA3.1/GSK3β/V5/His) and treated with 50 μM capsaicin for 6 h. The cell lysates were immunoprecipitated with ProBond nickel-chelating resin (Invitrogen), separated by sodium dodecyl sulfate–polyacrylamide gel, transferred to membranes, and then immunoblotted with V5, C/EBPβ or ATF3 antibody. (G) Chromatin immunoprecipitation assay for ATF3 binding was performed using a DNA–protein complex treated with 50 μM capsaicin for 24 h as described in Materials and Methods. The sequence of the NAG-1 promoter region (−131/+137) was amplified by PCR primer pairs as indicated by the arrows. The input represents PCR products obtained from 1% aliquots of chromatin pellets escaping immunoprecipitation.

    Techniques Used: Expressing, Western Blot, Isolation, Transfection, Plasmid Preparation, Immunoprecipitation, Chromatin Immunoprecipitation, Binding Assay, Sequencing, Amplification, Polymerase Chain Reaction

    Knockdown of C/EBPβ alters capsaicin-induced NAG-1 expression and apoptosis. (A) HCT-116 cells were transfected with control or C/EBPβ shRNA using Lipofectamine 2000 for 48 h and then treated with vehicle or 50 μM capsaicin for 24 h. Western analysis was performed for C/EBPβ, NAG-1, and actin. (B) HCT-116 cells were transfected with control or C/EBPβ shRNA and treated with 100 μM of capsaicin for 24 h. Apoptosis was analyzed as described in Materials and Methods. Values are expressed as mean ± SD of three replicates. (C) HCT-116 cells were transfected with control or ATF3 siRNA (200 nM) using TransIT-TKO Mirus transfection reagent for 48 h and then treated with ethanol or 50 μM of capsaicin for 24 h. Western analysis was performed for ATF3, NAG-1, and actin. (D) Proposed mechanism by which capsaicin induces NAG-1 transcription in human colorectal cancer. Capsaicin activates C/EBPβ through GSK3β- and PKCδ-dependent phosphorylation. Capsaicin increase nuclear accumulation of GSK3β and phosphorylates C/EBPβ through direct interaction with C/EBPβ. Activation of C/EBPβ increases the binding affinity of C/EBPβ onto NAG-1 promoter and activates transcription of NAG-1 genes. ATF3 may play a role as a bridge protein or formation of supracomplex, including ATF3 and other transcription factors, thereby enhancing the recruitment of C/EBPβ to the NAG-1 promoter. Upregulation of the NAG-1 gene results in an increase of apoptosis in colorectal cancer cells.
    Figure Legend Snippet: Knockdown of C/EBPβ alters capsaicin-induced NAG-1 expression and apoptosis. (A) HCT-116 cells were transfected with control or C/EBPβ shRNA using Lipofectamine 2000 for 48 h and then treated with vehicle or 50 μM capsaicin for 24 h. Western analysis was performed for C/EBPβ, NAG-1, and actin. (B) HCT-116 cells were transfected with control or C/EBPβ shRNA and treated with 100 μM of capsaicin for 24 h. Apoptosis was analyzed as described in Materials and Methods. Values are expressed as mean ± SD of three replicates. (C) HCT-116 cells were transfected with control or ATF3 siRNA (200 nM) using TransIT-TKO Mirus transfection reagent for 48 h and then treated with ethanol or 50 μM of capsaicin for 24 h. Western analysis was performed for ATF3, NAG-1, and actin. (D) Proposed mechanism by which capsaicin induces NAG-1 transcription in human colorectal cancer. Capsaicin activates C/EBPβ through GSK3β- and PKCδ-dependent phosphorylation. Capsaicin increase nuclear accumulation of GSK3β and phosphorylates C/EBPβ through direct interaction with C/EBPβ. Activation of C/EBPβ increases the binding affinity of C/EBPβ onto NAG-1 promoter and activates transcription of NAG-1 genes. ATF3 may play a role as a bridge protein or formation of supracomplex, including ATF3 and other transcription factors, thereby enhancing the recruitment of C/EBPβ to the NAG-1 promoter. Upregulation of the NAG-1 gene results in an increase of apoptosis in colorectal cancer cells.

    Techniques Used: Expressing, Transfection, shRNA, Western Blot, Activation Assay, Binding Assay

    29) Product Images from "Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity"

    Article Title: Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-7-809

    PCR amplification of the ureG gene and verification of the recombinant prokaryotic expression plasmid pRSET- ureG. A : PCR amplification of the ureG gene B : PCR screening of the pRSET/ ureG recombinant C : Digestion product of pRSET/ ureG with Hin dIII and Eco RI M : 100 bp DNA ladder.
    Figure Legend Snippet: PCR amplification of the ureG gene and verification of the recombinant prokaryotic expression plasmid pRSET- ureG. A : PCR amplification of the ureG gene B : PCR screening of the pRSET/ ureG recombinant C : Digestion product of pRSET/ ureG with Hin dIII and Eco RI M : 100 bp DNA ladder.

    Techniques Used: Polymerase Chain Reaction, Amplification, Recombinant, Expressing, Plasmid Preparation

    30) Product Images from "Clonotypic Analysis of Immunoglobulin Heavy Chain Sequences in Patients with Waldenström's Macroglobulinemia: Correlation with MYD88 L265P Somatic Mutation Status, Clinical Features, and Outcome"

    Article Title: Clonotypic Analysis of Immunoglobulin Heavy Chain Sequences in Patients with Waldenström's Macroglobulinemia: Correlation with MYD88 L265P Somatic Mutation Status, Clinical Features, and Outcome

    Journal: BioMed Research International

    doi: 10.1155/2014/809103

    Electropherogram—after capillary electrophoresis in Agilent 2100 Bioanalyzer using Agilent DNA 1000 kit (Agilent Technologies)—of IGHV6 -PCR product in one of the two patients of whom genomic DNA was extracted from blood sample and not bone marrow. Monoclonality of IGHV -PCR product is obvious (peak number 2) and was further confirmed by direct sequencing.
    Figure Legend Snippet: Electropherogram—after capillary electrophoresis in Agilent 2100 Bioanalyzer using Agilent DNA 1000 kit (Agilent Technologies)—of IGHV6 -PCR product in one of the two patients of whom genomic DNA was extracted from blood sample and not bone marrow. Monoclonality of IGHV -PCR product is obvious (peak number 2) and was further confirmed by direct sequencing.

    Techniques Used: Electrophoresis, Polymerase Chain Reaction, Sequencing

    31) Product Images from "RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs"

    Article Title: RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs

    Journal: Molecular and biochemical parasitology

    doi: 10.1016/j.molbiopara.2009.05.002

    Knockdown of egg hatching by RNA interference in Schistosoma mansoni . Hatching of eggs was blocked by dsRNA against Sm LAP1, Sm LAP2, both together, and bestatin. A. Representative micrographs of eggs during the hatching process, one hour after incubation in water. A: mock control, B: dsLUC treated eggs, C: dsLAP1 treated eggs, D: dsLAP2 treated eggs, E: eggs treated with dsLAP1 and dsLAP2, F: bestatin treated eggs. Bar, 200 μm. B. Percentage of hatched eggs: 100%, control eggs. Statistically significant differences were evident among the dsLAP1 group, dsLAP2 group, dsLAP1+dsLAP2 group, bestatin group and the controls mock and irrelevant dsRNA. The bars indicate standard deviation of mean for two experiments, and asterisks indicate significant differences ( P
    Figure Legend Snippet: Knockdown of egg hatching by RNA interference in Schistosoma mansoni . Hatching of eggs was blocked by dsRNA against Sm LAP1, Sm LAP2, both together, and bestatin. A. Representative micrographs of eggs during the hatching process, one hour after incubation in water. A: mock control, B: dsLUC treated eggs, C: dsLAP1 treated eggs, D: dsLAP2 treated eggs, E: eggs treated with dsLAP1 and dsLAP2, F: bestatin treated eggs. Bar, 200 μm. B. Percentage of hatched eggs: 100%, control eggs. Statistically significant differences were evident among the dsLAP1 group, dsLAP2 group, dsLAP1+dsLAP2 group, bestatin group and the controls mock and irrelevant dsRNA. The bars indicate standard deviation of mean for two experiments, and asterisks indicate significant differences ( P

    Techniques Used: Incubation, Standard Deviation

    Developmental stage specific expression of leucine aminopeptidases (LAP) in Schistosoma mansoni. Panel A. cDNAs from eggs (E), miracidia (M), sporocysts (Sp), cercariae (C), schistosomules (So), mix sex adults (A), male adults (♂) and female adults (♀) were employed as templates for PCRs using primers specific for the Sm LAP1, Sm LAP2 and S. mansoni actin genes. Panel B. Densitometric measurements (arbitrary units) across the different bands and relative levels of Sm LAP1 (blue) and Sm LAP2 (red) compared with levels for actin presented in panel A.
    Figure Legend Snippet: Developmental stage specific expression of leucine aminopeptidases (LAP) in Schistosoma mansoni. Panel A. cDNAs from eggs (E), miracidia (M), sporocysts (Sp), cercariae (C), schistosomules (So), mix sex adults (A), male adults (♂) and female adults (♀) were employed as templates for PCRs using primers specific for the Sm LAP1, Sm LAP2 and S. mansoni actin genes. Panel B. Densitometric measurements (arbitrary units) across the different bands and relative levels of Sm LAP1 (blue) and Sm LAP2 (red) compared with levels for actin presented in panel A.

    Techniques Used: Expressing

    RNAi targeting schistosome leucine aminopeptidases results in statistically significant knock down of protease activities and mRNA levels. Panel A. Leucine aminopeptidase (LAP) activity assay performed using the diagnostic substrate, L-leucine-7-amido-4-methylcoumarin hydroxide. LAP activity in relative fluorescence units per microgram (RLU/μg) of soluble schistosome protein presented on Y-axis. Groups of eggs were treated with double stranded RNA targeting Sm LAP1, Sm LAP2, both, firefly luciferase, and not treated with dsRNA (mock control). Panel B. Semi-quantitative RT-PCR using LAP specific primers and actin as an internal control. Schistosome egg cDNAs were synthesized from serial dilutions of total RNAs (100 ng, 10 ng, 1 ng) isolated from treatment group of eggs, soaked in doubled strand RNA targeting Sm LAP1, Sm LAP2, both, firefly luciferase, and not treated with dsRNA (mock control). The bars show the standard deviations for two experiments. Asterisks indicate significant differences ( P
    Figure Legend Snippet: RNAi targeting schistosome leucine aminopeptidases results in statistically significant knock down of protease activities and mRNA levels. Panel A. Leucine aminopeptidase (LAP) activity assay performed using the diagnostic substrate, L-leucine-7-amido-4-methylcoumarin hydroxide. LAP activity in relative fluorescence units per microgram (RLU/μg) of soluble schistosome protein presented on Y-axis. Groups of eggs were treated with double stranded RNA targeting Sm LAP1, Sm LAP2, both, firefly luciferase, and not treated with dsRNA (mock control). Panel B. Semi-quantitative RT-PCR using LAP specific primers and actin as an internal control. Schistosome egg cDNAs were synthesized from serial dilutions of total RNAs (100 ng, 10 ng, 1 ng) isolated from treatment group of eggs, soaked in doubled strand RNA targeting Sm LAP1, Sm LAP2, both, firefly luciferase, and not treated with dsRNA (mock control). The bars show the standard deviations for two experiments. Asterisks indicate significant differences ( P

    Techniques Used: Activity Assay, Diagnostic Assay, Fluorescence, Luciferase, Quantitative RT-PCR, Synthesized, Isolation

    A schematic view of the genome loci encoding the Schistosoma mansoni , indicates the position of the exons of Sm LAP1 and Sm LAP2 (coding portions in light blue boxes, 3′ untranslated regions in white boxes). High scoring regions on tblastx comparisons against the S. japonicum ] on the genomic sequence and their orientations are indicated.
    Figure Legend Snippet: A schematic view of the genome loci encoding the Schistosoma mansoni , indicates the position of the exons of Sm LAP1 and Sm LAP2 (coding portions in light blue boxes, 3′ untranslated regions in white boxes). High scoring regions on tblastx comparisons against the S. japonicum ] on the genomic sequence and their orientations are indicated.

    Techniques Used: Sequencing

    32) Product Images from "Small-Molecule Inhibitors Targeting Topoisomerase I as Novel Antituberculosis Agents"

    Article Title: Small-Molecule Inhibitors Targeting Topoisomerase I as Novel Antituberculosis Agents

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00288-16

    Effect of MtbTopI overexpression on the bactericidal effect of selected topoisomerase I inhibitors. The minimal bactericidal concentration (MBC) values against M. smegmatis mc 2 155 are shown in the table. The loss of viability following treatment with compounds for 44 h was compared between transformants overexpressing MtbTopI (M+) from pTA-M + and with control vector pTA-nol (nol). The downward arrow indicates that no viable colonies could be detected following 44 h of treatment with compound 2471-12 at a 50 μM concentration.
    Figure Legend Snippet: Effect of MtbTopI overexpression on the bactericidal effect of selected topoisomerase I inhibitors. The minimal bactericidal concentration (MBC) values against M. smegmatis mc 2 155 are shown in the table. The loss of viability following treatment with compounds for 44 h was compared between transformants overexpressing MtbTopI (M+) from pTA-M + and with control vector pTA-nol (nol). The downward arrow indicates that no viable colonies could be detected following 44 h of treatment with compound 2471-12 at a 50 μM concentration.

    Techniques Used: Over Expression, Concentration Assay, Plasmid Preparation

    33) Product Images from "R-Ras-Akt axis induces endothelial lumenogenesis and regulates the patency of regenerating vasculature"

    Article Title: R-Ras-Akt axis induces endothelial lumenogenesis and regulates the patency of regenerating vasculature

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01865-x

    RRAS gene delivery to ECs rescues vessel lumenogenesis and muscle reperfusion. a In vivo transduction and treatment schedule. The lentivirus carrying pLenti6/ Cdh5 -R-Ras38V expression vector was injected into GC muscles 3 days after ligation. GC muscle were analyzed at day 14. b Immunofluorescence of CD31 and PODXL to identify lumenized vessels in GC muscles after the lentivirus injection for EC-specific expression of R-Ras38V (R-Ras38V EC ). PODXL positivity % (PODXL + CD31 + area/CD31 + area × 100) was determined to assess the fraction of lumenized vessel area. c Transmission electron microscopy of the GC muscles confirmed the increase in vessel lumenization upon R-Ras38V EC transduction. Arrow, a circulating erythrocyte found in the vessel lumen indicating normal lumen formation. d CD31 staining of whole-mounted GC muscle fascicles. e Analysis of vessel perfusion in whole-mounted GC muscle fascicles. Yellow color indicates lectin perfused vessels. f H E staining of GC muscle sections. Arrows, necrotic areas. g Dystrophin immunostaining (green) of GC muscle cross-sections to quantify functional muscle fibers. The number of dystrophin + muscle fibers/total muscle fibers (%) was determined in non-necrotic area. p
    Figure Legend Snippet: RRAS gene delivery to ECs rescues vessel lumenogenesis and muscle reperfusion. a In vivo transduction and treatment schedule. The lentivirus carrying pLenti6/ Cdh5 -R-Ras38V expression vector was injected into GC muscles 3 days after ligation. GC muscle were analyzed at day 14. b Immunofluorescence of CD31 and PODXL to identify lumenized vessels in GC muscles after the lentivirus injection for EC-specific expression of R-Ras38V (R-Ras38V EC ). PODXL positivity % (PODXL + CD31 + area/CD31 + area × 100) was determined to assess the fraction of lumenized vessel area. c Transmission electron microscopy of the GC muscles confirmed the increase in vessel lumenization upon R-Ras38V EC transduction. Arrow, a circulating erythrocyte found in the vessel lumen indicating normal lumen formation. d CD31 staining of whole-mounted GC muscle fascicles. e Analysis of vessel perfusion in whole-mounted GC muscle fascicles. Yellow color indicates lectin perfused vessels. f H E staining of GC muscle sections. Arrows, necrotic areas. g Dystrophin immunostaining (green) of GC muscle cross-sections to quantify functional muscle fibers. The number of dystrophin + muscle fibers/total muscle fibers (%) was determined in non-necrotic area. p

    Techniques Used: In Vivo, Transduction, Expressing, Plasmid Preparation, Injection, Ligation, Immunofluorescence, Transmission Assay, Electron Microscopy, Staining, Immunostaining, Functional Assay

    34) Product Images from "Genetic targeting of the endoderm with claudin-6CreER"

    Article Title: Genetic targeting of the endoderm with claudin-6CreER

    Journal: Developmental Dynamics

    doi: 10.1002/dvdy.21437

    Successful targeting of the Cldn6 locus with the CIHV cassette. A: Southern blot analysis. Upon successful recombination, the entire coding sequence for Cldn6 is removed, generating a null allele. After NcoI digest, the wild type locus generates a band of approximately 12.5 kb, while the targeted allele has a smaller band close to 8 kb. For reference, the first two bands on the 1-kb ladder are 10 and 8 kb, respectively. As seen in the parental ES cell lane (AV3), only one band is present higher than 10 kb, while in the targeted ES cell clone lane (E40), there is a band at the same height as in the wild type lane, but a second band present at 8 kb. B: Genotyping by PCR confirms germline transmission of the CIHV allele. AV3 and E40 refer to the parental ES cell line and targeted ES cell line used for blastocyst injection, respectively. The upper gel distinguishes between the wild type and targeted locus. The lower gel is a PCR to demonstrate ACN cassette removal during germline transmission. C,D: Cldn6 CIHV/CIHV mice are null mutants for Cldn6 . In situ hybridization for Cldn6 in embryos from a Cldn6 CIHV/+ intercross. C: Cldn6 CIHV/+ ; D: Cldn6 CIHV/CIHV . As shown in D, Cldn6 mRNA is absent in the Cldn6 CIHV/CIHV embryo.
    Figure Legend Snippet: Successful targeting of the Cldn6 locus with the CIHV cassette. A: Southern blot analysis. Upon successful recombination, the entire coding sequence for Cldn6 is removed, generating a null allele. After NcoI digest, the wild type locus generates a band of approximately 12.5 kb, while the targeted allele has a smaller band close to 8 kb. For reference, the first two bands on the 1-kb ladder are 10 and 8 kb, respectively. As seen in the parental ES cell lane (AV3), only one band is present higher than 10 kb, while in the targeted ES cell clone lane (E40), there is a band at the same height as in the wild type lane, but a second band present at 8 kb. B: Genotyping by PCR confirms germline transmission of the CIHV allele. AV3 and E40 refer to the parental ES cell line and targeted ES cell line used for blastocyst injection, respectively. The upper gel distinguishes between the wild type and targeted locus. The lower gel is a PCR to demonstrate ACN cassette removal during germline transmission. C,D: Cldn6 CIHV/CIHV mice are null mutants for Cldn6 . In situ hybridization for Cldn6 in embryos from a Cldn6 CIHV/+ intercross. C: Cldn6 CIHV/+ ; D: Cldn6 CIHV/CIHV . As shown in D, Cldn6 mRNA is absent in the Cldn6 CIHV/CIHV embryo.

    Techniques Used: Southern Blot, Sequencing, Polymerase Chain Reaction, Transmission Assay, Injection, Mouse Assay, In Situ Hybridization

    35) Product Images from "Cloning, Expression, and Functional Analysis of Rat Liver Cytosolic Inorganic Pyrophosphatase Gene and Characterization of its Functional Promoter"

    Article Title: Cloning, Expression, and Functional Analysis of Rat Liver Cytosolic Inorganic Pyrophosphatase Gene and Characterization of its Functional Promoter

    Journal: Gene Expression

    doi:

    Western blot analysis of expressed His-tagged rat liver cytosolic inorganic pyrophosphatase using anti-His-HRP-conjugated antibody. Whole cell lysate of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA ; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 6, Ni-NTA purified recombinant rat liver cytosolic iPPase protein by denaturing method. In each lane 10 μg of protein was taken except for lane 6, where 5 μg purified recombinant protein was used.
    Figure Legend Snippet: Western blot analysis of expressed His-tagged rat liver cytosolic inorganic pyrophosphatase using anti-His-HRP-conjugated antibody. Whole cell lysate of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA ; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 6, Ni-NTA purified recombinant rat liver cytosolic iPPase protein by denaturing method. In each lane 10 μg of protein was taken except for lane 6, where 5 μg purified recombinant protein was used.

    Techniques Used: Western Blot, Transformation Assay, Expressing, Plasmid Preparation, Purification, Recombinant

    Study of functional activity of expressed rat liver cytosolic iPPase by in-gel assay. Whole cell lysate protein (12 μg) of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector. Lane 6: 5 μg of Ni-NTA column purified renatured rat liver cytosolic iPPase. Lane 7: 40 μg of rat liver WCL. Lanes 1–4 show only endogenous iPPase activity of E. coli. Lane 5 shows activity of expressed rat liver iPPase as well as endogenous E. coli iPPase. Lane 6 shows activity of purified bacterially expressed rat liver iPPase. Lane 7 shows native rat liver iPPase.
    Figure Legend Snippet: Study of functional activity of expressed rat liver cytosolic iPPase by in-gel assay. Whole cell lysate protein (12 μg) of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector. Lane 6: 5 μg of Ni-NTA column purified renatured rat liver cytosolic iPPase. Lane 7: 40 μg of rat liver WCL. Lanes 1–4 show only endogenous iPPase activity of E. coli. Lane 5 shows activity of expressed rat liver iPPase as well as endogenous E. coli iPPase. Lane 6 shows activity of purified bacterially expressed rat liver iPPase. Lane 7 shows native rat liver iPPase.

    Techniques Used: Functional Assay, Activity Assay, Transformation Assay, Expressing, Plasmid Preparation, Purification

    SDS-PAGE analysis of expressed His-tagged rat liver cytosolic inorganic pyrophosphatase. Lane M: molecular weight marker in kDa. Whole cell lysate of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA ; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 6, Ni-NTA purified recombinant rat liver cytosolic iPPase protein by denaturing method. In each lane 10 μg of protein was taken except for lane 6, where 5 μg purified recombinant protein was used.
    Figure Legend Snippet: SDS-PAGE analysis of expressed His-tagged rat liver cytosolic inorganic pyrophosphatase. Lane M: molecular weight marker in kDa. Whole cell lysate of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA ; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 6, Ni-NTA purified recombinant rat liver cytosolic iPPase protein by denaturing method. In each lane 10 μg of protein was taken except for lane 6, where 5 μg purified recombinant protein was used.

    Techniques Used: SDS Page, Molecular Weight, Marker, Transformation Assay, Expressing, Plasmid Preparation, Purification, Recombinant

    36) Product Images from "Expression of C-terminal deleted p53 isoforms in neuroblastoma"

    Article Title: Expression of C-terminal deleted p53 isoforms in neuroblastoma

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl619

    p53 transactivation ability by luciferase test using plasmid pE1B-hWAF1( A ) and Bax ( B ). p53-deficient LAN-1 cells (p53-) (left panel) or SH-SY5Y (p53+) (right panel) were cotransfected with 0.5 μg of the luciferase reporter gene containing the human p21/WAF1 -p53-responsive element and with 1 μg of the expressing vector as indicated. Cells were collected and subject to luciferase assay, 24 h following cotransfection. The values represent mean relative luciferase activity from three independent experiments. SH-SY5Y, IGR-N-91, SK-N-BE(2), SK-N-AS and IGR-NB8 were termed as SH, N91, BE(2), AS and NB8, respectively.
    Figure Legend Snippet: p53 transactivation ability by luciferase test using plasmid pE1B-hWAF1( A ) and Bax ( B ). p53-deficient LAN-1 cells (p53-) (left panel) or SH-SY5Y (p53+) (right panel) were cotransfected with 0.5 μg of the luciferase reporter gene containing the human p21/WAF1 -p53-responsive element and with 1 μg of the expressing vector as indicated. Cells were collected and subject to luciferase assay, 24 h following cotransfection. The values represent mean relative luciferase activity from three independent experiments. SH-SY5Y, IGR-N-91, SK-N-BE(2), SK-N-AS and IGR-NB8 were termed as SH, N91, BE(2), AS and NB8, respectively.

    Techniques Used: Luciferase, Plasmid Preparation, Expressing, Cotransfection, Activity Assay

    Detection of p53 mRNA abnormalities in SK-N-AS (AS) in comparison with SH-SY5Y (SH) cells. ( A ) Amplification of p53 cDNA using primers from exons 8 to 10 as indicated below each arrow; for precise position see Table 1 and GenBank K03199: F2 (forward primer in exon 8); R3 (reverse primer at the junction of exon 8/9); R4 (the first moiety of exon 9); R5 (exon 9, 30 nt downstream R4); R6 (beginning of exon 10). Note that no amplification was observed in SK-N-AS from exon 10 (last lane), in contrast to SH-SY5Y. ( B ) RT–PCR from SK-N-AS compared to SH-SY5Y cells. Specific primers ( Table 1 ) were used to amplify the DBD, the p53β isoform (β) and the C-terminal domain (C-ter).
    Figure Legend Snippet: Detection of p53 mRNA abnormalities in SK-N-AS (AS) in comparison with SH-SY5Y (SH) cells. ( A ) Amplification of p53 cDNA using primers from exons 8 to 10 as indicated below each arrow; for precise position see Table 1 and GenBank K03199: F2 (forward primer in exon 8); R3 (reverse primer at the junction of exon 8/9); R4 (the first moiety of exon 9); R5 (exon 9, 30 nt downstream R4); R6 (beginning of exon 10). Note that no amplification was observed in SK-N-AS from exon 10 (last lane), in contrast to SH-SY5Y. ( B ) RT–PCR from SK-N-AS compared to SH-SY5Y cells. Specific primers ( Table 1 ) were used to amplify the DBD, the p53β isoform (β) and the C-terminal domain (C-ter).

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction

    Structure of p53 proteins in different neuroblastoma cell lines. The three functional domains are represented: TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. The wild-type p53 gene in SH-SY5Y, IMR-32 and LAN-5 cells contains 11 exons that encode 393 amino acids. In SK-N-BE(2) cells, p53 is mutated at codon 135 ( * ), which converts cysteine to phenylalanine. In IGR-N-91 cells, a duplication of exons 7-8-9 adds an additional 107 amino acids leading to a total of 500. In SK-N-AS cells, a mutation due to alternate splicing downstream of exon 9 leads to a protein of 341 amino acids whereas in IGR-NB8 cells, the p53 protein ends at 326 amino acids owing to the mutation E326STOP.
    Figure Legend Snippet: Structure of p53 proteins in different neuroblastoma cell lines. The three functional domains are represented: TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. The wild-type p53 gene in SH-SY5Y, IMR-32 and LAN-5 cells contains 11 exons that encode 393 amino acids. In SK-N-BE(2) cells, p53 is mutated at codon 135 ( * ), which converts cysteine to phenylalanine. In IGR-N-91 cells, a duplication of exons 7-8-9 adds an additional 107 amino acids leading to a total of 500. In SK-N-AS cells, a mutation due to alternate splicing downstream of exon 9 leads to a protein of 341 amino acids whereas in IGR-NB8 cells, the p53 protein ends at 326 amino acids owing to the mutation E326STOP.

    Techniques Used: Functional Assay, Binding Assay, Mutagenesis

    Identification of a deletion spanning the intron9/exon 10 junction of SK-N-AS p53 gene. ( A ) Schematic representation of p53 gene from intron 7 to intron 10 with the position of amplified fragments; ( B ) PCR fragments amplified from SH-SY5Y (SH) and SK-N-AS (AS) DNA with the primer pairs a, b, c and d. The primer sequences are given in Table 1 ; contl: PCR performed in parallel without DNA.
    Figure Legend Snippet: Identification of a deletion spanning the intron9/exon 10 junction of SK-N-AS p53 gene. ( A ) Schematic representation of p53 gene from intron 7 to intron 10 with the position of amplified fragments; ( B ) PCR fragments amplified from SH-SY5Y (SH) and SK-N-AS (AS) DNA with the primer pairs a, b, c and d. The primer sequences are given in Table 1 ; contl: PCR performed in parallel without DNA.

    Techniques Used: Amplification, Polymerase Chain Reaction

    37) Product Images from "sMEK1 inhibits endothelial cell proliferation by attenuating VEGFR-2-dependent-Akt/eNOS/HIF-1α signaling pathways"

    Article Title: sMEK1 inhibits endothelial cell proliferation by attenuating VEGFR-2-dependent-Akt/eNOS/HIF-1α signaling pathways

    Journal: Oncotarget

    doi:

    sMEK1 suppressed the phosphorylated proteins of the VEGFR-2/PI3K/eNOS signaling cascade Cell lysates were prepared from transfected SKOV-3 cancer cells or HUVECs (data not shown) and subjected to immunoblotting using primary antibodies specific to the phosphorylated or unphosphorylated forms of negative and positive regulators of Akt, including PI3K and eNOS. All experiments were repeated at least three times with similar results.
    Figure Legend Snippet: sMEK1 suppressed the phosphorylated proteins of the VEGFR-2/PI3K/eNOS signaling cascade Cell lysates were prepared from transfected SKOV-3 cancer cells or HUVECs (data not shown) and subjected to immunoblotting using primary antibodies specific to the phosphorylated or unphosphorylated forms of negative and positive regulators of Akt, including PI3K and eNOS. All experiments were repeated at least three times with similar results.

    Techniques Used: Transfection

    sMEK1 decreases VEGF-stimulated VEGFR-2 phosphorylation (Tyr-951) A. HUVECs were transfected with different concentrations of sMEK1, and total cell lysates were prepared. The expression of total and phosphorylated (Tyr-951 or Tyr-1175) VEGFR-2 was measured by immunoblot analysis in cells treated with VEGF. B. HUVECs were treated with VEGF and then transfected with the control vector, sMEK1, or sMEK1 plus sisMEK1. sMEK1 suppressed VEGF-stimulated VEGFR-2 (Tyr-951) phosphorylation. VEGFR-2 (Tyr-951 or Tyr-1175) phosphorylation was evaluated using specific antibodies, and VEGFR-2 was used as a loading control. C. sMEK1 suppressed VEGFR-2 kinase activity in vitro , analyzed using an in vitro HTScan VEGFR-2 kinase assay kit followed by colorimetric detection according to the manufacturer's protocols. Values are presented as means±SDs; *, P
    Figure Legend Snippet: sMEK1 decreases VEGF-stimulated VEGFR-2 phosphorylation (Tyr-951) A. HUVECs were transfected with different concentrations of sMEK1, and total cell lysates were prepared. The expression of total and phosphorylated (Tyr-951 or Tyr-1175) VEGFR-2 was measured by immunoblot analysis in cells treated with VEGF. B. HUVECs were treated with VEGF and then transfected with the control vector, sMEK1, or sMEK1 plus sisMEK1. sMEK1 suppressed VEGF-stimulated VEGFR-2 (Tyr-951) phosphorylation. VEGFR-2 (Tyr-951 or Tyr-1175) phosphorylation was evaluated using specific antibodies, and VEGFR-2 was used as a loading control. C. sMEK1 suppressed VEGFR-2 kinase activity in vitro , analyzed using an in vitro HTScan VEGFR-2 kinase assay kit followed by colorimetric detection according to the manufacturer's protocols. Values are presented as means±SDs; *, P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Activity Assay, In Vitro, Kinase Assay

    Physical interaction between sMEK1 and VEGFR-2 A. Positive interactions were confirmed by observed cell growth on a medium lacking leucine and by the formation of blue colonies on X-gal plates containing 2% galactose. β-galactosidase activity (unit), measured by adding o -nitrophenyl β- D -galactopyranoside (ONPG), is shown below the corresponding lanes. B. The co-immunoprecipitation of sMEK1 with VEGFR-1 or VEGFR-2. Immunoprecipitation from transfected HEK293T cells was performed using anti-FLAG antibodies in lysates, followed by immunoblotting with anti-sMEK1, anti-VEGFR-1, and anti-VEGFR-2 antibodies. C. Endogenous proteins in total lysates of HEK293T cells were subjected to co-immunoprecipitation (IP) with an antibody as indicated followed by Western blotting (WB) with an anti-sMEK1 or anti-VEGFR-2 antibody. A rabbit IgG and VEGFR-1 were included as an IP negative control. The input (Non-IP) WB data indicated the integrity of lysates used for IP. D. Mapping of the VEGFR-2 region critical for the interaction with sMEK1 using a protein-protein interaction assay in vivo . The cDNA constructs were co-transformed into EGY48 yeast cells, and protein-protein interactions were assessed using a yeast two-hybrid system. Positive interactions were confirmed by observed cell growth (upper panel) and the formation of blue colonies (lower panel). The β-galactosidase activity of each construct in negative controls (vector only) was
    Figure Legend Snippet: Physical interaction between sMEK1 and VEGFR-2 A. Positive interactions were confirmed by observed cell growth on a medium lacking leucine and by the formation of blue colonies on X-gal plates containing 2% galactose. β-galactosidase activity (unit), measured by adding o -nitrophenyl β- D -galactopyranoside (ONPG), is shown below the corresponding lanes. B. The co-immunoprecipitation of sMEK1 with VEGFR-1 or VEGFR-2. Immunoprecipitation from transfected HEK293T cells was performed using anti-FLAG antibodies in lysates, followed by immunoblotting with anti-sMEK1, anti-VEGFR-1, and anti-VEGFR-2 antibodies. C. Endogenous proteins in total lysates of HEK293T cells were subjected to co-immunoprecipitation (IP) with an antibody as indicated followed by Western blotting (WB) with an anti-sMEK1 or anti-VEGFR-2 antibody. A rabbit IgG and VEGFR-1 were included as an IP negative control. The input (Non-IP) WB data indicated the integrity of lysates used for IP. D. Mapping of the VEGFR-2 region critical for the interaction with sMEK1 using a protein-protein interaction assay in vivo . The cDNA constructs were co-transformed into EGY48 yeast cells, and protein-protein interactions were assessed using a yeast two-hybrid system. Positive interactions were confirmed by observed cell growth (upper panel) and the formation of blue colonies (lower panel). The β-galactosidase activity of each construct in negative controls (vector only) was

    Techniques Used: Activity Assay, Immunoprecipitation, Transfection, Western Blot, Negative Control, Protein Protein Interaction Assay, In Vivo, Construct, Transformation Assay, Plasmid Preparation

    38) Product Images from "Increased expression of transcription factor TFAP2? correlates with chemosensitivity in advanced bladder cancer"

    Article Title: Increased expression of transcription factor TFAP2? correlates with chemosensitivity in advanced bladder cancer

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-11-135

    Expression of TFAP2α isoforms . (A) Expression of TFAP2α isoform 1, 2 and 3 in advanced muscle invasive bladder cancer (T2-4) were determined using real-time Q-PCR. Analysis was performed on cDNA from 10 tumor specimens and each bar represents the mean from the 10 samples.(B) COS-7 cells were transiently transfected with empty pcDNA3.1/V5-His vector (lane 2, 6), pcDNA3.1/V5-His- TFAP2α isoform 1(lane 3, 7), isoform 2 (lane 4, 8) and isoform 3 (lane 5, 9). Western blot of 30 μg total protein lysate from non-transfected HU609 bladder cells (lane 1) and COS-7 transfected cells (lane 2-9) 48 h post transfection probed with anti TFAP2α antibody (lane 1-5) or anti-V5 antibody (lane 6-9).
    Figure Legend Snippet: Expression of TFAP2α isoforms . (A) Expression of TFAP2α isoform 1, 2 and 3 in advanced muscle invasive bladder cancer (T2-4) were determined using real-time Q-PCR. Analysis was performed on cDNA from 10 tumor specimens and each bar represents the mean from the 10 samples.(B) COS-7 cells were transiently transfected with empty pcDNA3.1/V5-His vector (lane 2, 6), pcDNA3.1/V5-His- TFAP2α isoform 1(lane 3, 7), isoform 2 (lane 4, 8) and isoform 3 (lane 5, 9). Western blot of 30 μg total protein lysate from non-transfected HU609 bladder cells (lane 1) and COS-7 transfected cells (lane 2-9) 48 h post transfection probed with anti TFAP2α antibody (lane 1-5) or anti-V5 antibody (lane 6-9).

    Techniques Used: Expressing, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot

    39) Product Images from "Mutations in an Arabidopsis Mitochondrial Transcription Termination Factor-Related Protein Enhance Thermotolerance in the Absence of the Major Molecular Chaperone HSP101 [W]"

    Article Title: Mutations in an Arabidopsis Mitochondrial Transcription Termination Factor-Related Protein Enhance Thermotolerance in the Absence of the Major Molecular Chaperone HSP101 [W]

    Journal: The Plant Cell

    doi: 10.1105/tpc.112.101006

    The shot1-2 Mutant Can Suppress hot1-3 and Other Heat-Sensitive Mutants.
    Figure Legend Snippet: The shot1-2 Mutant Can Suppress hot1-3 and Other Heat-Sensitive Mutants.

    Techniques Used: Mutagenesis

    Organelle Gene Copy Numbers in shot1-2 .
    Figure Legend Snippet: Organelle Gene Copy Numbers in shot1-2 .

    Techniques Used:

    and AOXs Are Upregulated in the shot1-2 Mutant.
    Figure Legend Snippet: and AOXs Are Upregulated in the shot1-2 Mutant.

    Techniques Used: Mutagenesis

    The shot1-2 and Oxidative Damage and Is More Resistant to the Oxidative Agent Paraquat.
    Figure Legend Snippet: The shot1-2 and Oxidative Damage and Is More Resistant to the Oxidative Agent Paraquat.

    Techniques Used:

    Most Mitochondrion-Encoded Genes Are Upregulated and Plastid-Encoded Genes Are Downregulated in the shot1-2 Mutant.
    Figure Legend Snippet: Most Mitochondrion-Encoded Genes Are Upregulated and Plastid-Encoded Genes Are Downregulated in the shot1-2 Mutant.

    Techniques Used: Mutagenesis

    40) Product Images from "Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment"

    Article Title: Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00600-2

    Pair-wise mixed culture competition experiments. Competence assays of environmental strains Pseudomonas sp. 1SL5 and E . coli 4IgSN1, against the corresponding ARGC ( aadB or bla VIM-2 ) inserted at the native attI1 site containing strain were conducted in order to determine the relative fitness. Error bars indicate 95% confidence intervals. By definition, a relative fitness (w) of 1.00 indicates no difference in relative fitness. Relative fitness values: Pseudomonas sp. 1SL5:: aadB W = 1.03 (0.88–1.18), E . coli 4IgSN1:: aadB W = 1.02 (0.86–1.17), E . coli 4IgSN1:: bla VIM-2 W = 0.97 (0.92–1.01). CI is confidence interval.
    Figure Legend Snippet: Pair-wise mixed culture competition experiments. Competence assays of environmental strains Pseudomonas sp. 1SL5 and E . coli 4IgSN1, against the corresponding ARGC ( aadB or bla VIM-2 ) inserted at the native attI1 site containing strain were conducted in order to determine the relative fitness. Error bars indicate 95% confidence intervals. By definition, a relative fitness (w) of 1.00 indicates no difference in relative fitness. Relative fitness values: Pseudomonas sp. 1SL5:: aadB W = 1.03 (0.88–1.18), E . coli 4IgSN1:: aadB W = 1.02 (0.86–1.17), E . coli 4IgSN1:: bla VIM-2 W = 0.97 (0.92–1.01). CI is confidence interval.

    Techniques Used: Bla VIM Assay

    Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.
    Figure Legend Snippet: Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.

    Techniques Used: Isolation, Modification, Selection, Transformation Assay, Bla VIM Assay, Functional Assay, Plasmid Preparation

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    Sequencing:

    Article Title: Mechanism of PDK1-catalyzed Thr-229 Phosphorylation of the S6K1 Protein Kinase *
    Article Snippet: .. The catalytic kinase domains of PDK1 (His6 -PDK1(ΔPH), residues 51–359) and S6K1αII (native and T389E mutant His6 -S6K1αII(ΔAID), residues 1–398), each containing an N-terminal His6 tag (HHHHHH) followed by a PreScission protease recognition sequence (LEVLFQGP), were expressed using the Bac-to-Bac® baculovirus expression system (Invitrogen) and His6 affinity-purified as described ( ). .. The Thr-229-phosphorylated product forms of His6 -S6K1αII(ΔAID) were generated in the same manner except that (i) Sf9 insect cells were coinfected with recombinant baculovirus expressing His6 -PDK1(ΔPH), and (ii) His6 -S6K1αII(ΔAID) was resolved from His6 -PDK1(ΔPH) by MonoQ ion exchange chromatography as described ( ).

    Western Blot:

    Article Title: Coupled termination/reinitiation for translation of the downstream open reading frame B of the prototypic hypovirus CHV1-EP713
    Article Snippet: .. Baculovirus expression of reporter sequences and western blotting Reporter constructs for determination of ORF B translation efficiency were cloned into the baculovirus transfer vector pFastBacDual (Invitrogen). .. Reporter sequences on pFastBacDual were moved to baculovirus DNA retained as bacmid in DH10Bac Escherichia coli cells and then transfected into Spodoptera frugiperda cells (Sf9) according to the manufacturer's protocol (Bac-to-Bac Baculovirus Expression System, Invitrogen).

    Affinity Purification:

    Article Title: Mechanism of PDK1-catalyzed Thr-229 Phosphorylation of the S6K1 Protein Kinase *
    Article Snippet: .. The catalytic kinase domains of PDK1 (His6 -PDK1(ΔPH), residues 51–359) and S6K1αII (native and T389E mutant His6 -S6K1αII(ΔAID), residues 1–398), each containing an N-terminal His6 tag (HHHHHH) followed by a PreScission protease recognition sequence (LEVLFQGP), were expressed using the Bac-to-Bac® baculovirus expression system (Invitrogen) and His6 affinity-purified as described ( ). .. The Thr-229-phosphorylated product forms of His6 -S6K1αII(ΔAID) were generated in the same manner except that (i) Sf9 insect cells were coinfected with recombinant baculovirus expressing His6 -PDK1(ΔPH), and (ii) His6 -S6K1αII(ΔAID) was resolved from His6 -PDK1(ΔPH) by MonoQ ion exchange chromatography as described ( ).

    Recombinant:

    Article Title: Antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (VHSV) purified in large amounts from insect larvae
    Article Snippet: .. Recombinant baculoviruses were then constructed using the Bac-to-Bac® baculovirus expression system (Invitrogen) and following the manufacturer's instructions. .. The Bac-to-Bac site-specific transposition system uses the DH10 Bac™strain of E. Coli , which contains a Tn7 helper plasmid to insert exogenous Tn7-flanked genes into the Tn7 target site of its 150-kb bacmid DNA.

    Article Title: Recombinant H7 hemagglutinin forms subviral particles that protect mice and ferrets from challenge with H7N9 influenza virus
    Article Snippet: .. Recombinant BV expressing H7 HA gene was generated by using a Bac-to-Bac baculovirus expression system (Life Technologies). .. The presence of BV during virus stock preparation was monitored by Virus Counter (ViroCyt, Boulder, CO).

    Article Title: A time-resolved immunoassay to measure serum antibodies to the rotavirus VP6 capsid protein
    Article Snippet: .. Recombinant baculovirus (rBV) encoding these VP6 genes were produced using the Bac-to-Bac® baculovirus expression system (Invitrogen). .. The VP6 cDNA expression cassette was transferred from pDEST17 into the baculovirus shuttle DNA (bacmid) by transposition within chemically competent E. coli DH10Bac™ cells.

    Plasmid Preparation:

    Article Title: Coupled termination/reinitiation for translation of the downstream open reading frame B of the prototypic hypovirus CHV1-EP713
    Article Snippet: .. Baculovirus expression of reporter sequences and western blotting Reporter constructs for determination of ORF B translation efficiency were cloned into the baculovirus transfer vector pFastBacDual (Invitrogen). .. Reporter sequences on pFastBacDual were moved to baculovirus DNA retained as bacmid in DH10Bac Escherichia coli cells and then transfected into Spodoptera frugiperda cells (Sf9) according to the manufacturer's protocol (Bac-to-Bac Baculovirus Expression System, Invitrogen).

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