one hybrid expression vector encoding gal4dbd math6  (TaKaRa)

 
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    Structured Review

    TaKaRa one hybrid expression vector encoding gal4dbd math6
    <t>Math6</t> can function as a transcriptional repressor in vitro. A high background reporter plasmid consisting of five tandem copies of the Gal4 UAS upstream of the prolactin minimal promoter driving luciferase was cotransfected with increasing amounts of the plasmid expressing a fusion protein comprised of the GAL4 DNA binding domain <t>(GAL4DBD)</t> and full length Math6. Additionally, a GAL4DBD-Retinoblastoma Large Pocket (RbLP) and a GAL4-p300 (fragment 1737–2414 nt) fusion proteins were included as controls for repression and activation, respectively. Relative luciferase activities were calculated with the activity of cells transfected with the lowest amount of the GAL4DBD alone set at 1. All data are mean±SEM from transfections performed in duplicates on at least 4 occasions. * P
    One Hybrid Expression Vector Encoding Gal4dbd Math6, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 7545 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Identification of the bHLH Factor Math6 as a Novel Component of the Embryonic Pancreas Transcriptional Network"

    Article Title: Identification of the bHLH Factor Math6 as a Novel Component of the Embryonic Pancreas Transcriptional Network

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002430

    Math6 can function as a transcriptional repressor in vitro. A high background reporter plasmid consisting of five tandem copies of the Gal4 UAS upstream of the prolactin minimal promoter driving luciferase was cotransfected with increasing amounts of the plasmid expressing a fusion protein comprised of the GAL4 DNA binding domain (GAL4DBD) and full length Math6. Additionally, a GAL4DBD-Retinoblastoma Large Pocket (RbLP) and a GAL4-p300 (fragment 1737–2414 nt) fusion proteins were included as controls for repression and activation, respectively. Relative luciferase activities were calculated with the activity of cells transfected with the lowest amount of the GAL4DBD alone set at 1. All data are mean±SEM from transfections performed in duplicates on at least 4 occasions. * P
    Figure Legend Snippet: Math6 can function as a transcriptional repressor in vitro. A high background reporter plasmid consisting of five tandem copies of the Gal4 UAS upstream of the prolactin minimal promoter driving luciferase was cotransfected with increasing amounts of the plasmid expressing a fusion protein comprised of the GAL4 DNA binding domain (GAL4DBD) and full length Math6. Additionally, a GAL4DBD-Retinoblastoma Large Pocket (RbLP) and a GAL4-p300 (fragment 1737–2414 nt) fusion proteins were included as controls for repression and activation, respectively. Relative luciferase activities were calculated with the activity of cells transfected with the lowest amount of the GAL4DBD alone set at 1. All data are mean±SEM from transfections performed in duplicates on at least 4 occasions. * P

    Techniques Used: In Vitro, Plasmid Preparation, Luciferase, Expressing, Binding Assay, Activation Assay, Activity Assay, Transfection

    2) Product Images from "A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility"

    Article Title: A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility

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

    doi: 10.1073/pnas.1310777111

    Screening for mutant GALNTL5 in human male individuals. ( A ) Comparison of protein components in sperm from healthy volunteers (samples 1–4) and patients diagnosed with male infertility (samples 5–9). ( B ) DNA sequence chromatograms of the
    Figure Legend Snippet: Screening for mutant GALNTL5 in human male individuals. ( A ) Comparison of protein components in sperm from healthy volunteers (samples 1–4) and patients diagnosed with male infertility (samples 5–9). ( B ) DNA sequence chromatograms of the

    Techniques Used: Mutagenesis, Sequencing

    Male mouse sterility caused by heterozygous mutation of Galntl5 . ( A ) Fertility of wild-type (WT), heterozygous mutant (Ht) males, and Ht females. ( B and C ) Morphological phenotypes of epididymal spermatozoa from WT and Ht mice. Arrows indicate deformed
    Figure Legend Snippet: Male mouse sterility caused by heterozygous mutation of Galntl5 . ( A ) Fertility of wild-type (WT), heterozygous mutant (Ht) males, and Ht females. ( B and C ) Morphological phenotypes of epididymal spermatozoa from WT and Ht mice. Arrows indicate deformed

    Techniques Used: Sterility, Mutagenesis, Mouse Assay

    Localization of mouse GALNTL5 protein during spermiogenesis. Sections of adult mouse testis were immunostained with anti-GALNTL5 antibodies (red). The acrosomal vesicles and nuclei were counterstained with PNA (green) and DAPI (blue), respectively. (
    Figure Legend Snippet: Localization of mouse GALNTL5 protein during spermiogenesis. Sections of adult mouse testis were immunostained with anti-GALNTL5 antibodies (red). The acrosomal vesicles and nuclei were counterstained with PNA (green) and DAPI (blue), respectively. (

    Techniques Used:

    Heterozygous Mutation of Galntl5 Results in Immotile Sperm.
    Figure Legend Snippet: Heterozygous Mutation of Galntl5 Results in Immotile Sperm.

    Techniques Used: Mutagenesis

    3) Product Images from "SIMPLE/LITAF Expression Induces the Translocation of the Ubiquitin Ligase Itch towards the Lysosomal Compartments"

    Article Title: SIMPLE/LITAF Expression Induces the Translocation of the Ubiquitin Ligase Itch towards the Lysosomal Compartments

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016873

    LITAF changes the cellular localization of Itch. FLAG-LITAF was transiently co-transfected into BGMK cells with GFP-Itch. Sixteen hours post-transfection, cells were probed with LysoTracker (blue) and fixed. LITAF was detected using anti-FLAG antibodies (red) while the trans-Golgi network was identified using anti-IGF-IIR antibodies (blue). GFP-Itch is shown in green. Cells were visualized using DIC.
    Figure Legend Snippet: LITAF changes the cellular localization of Itch. FLAG-LITAF was transiently co-transfected into BGMK cells with GFP-Itch. Sixteen hours post-transfection, cells were probed with LysoTracker (blue) and fixed. LITAF was detected using anti-FLAG antibodies (red) while the trans-Golgi network was identified using anti-IGF-IIR antibodies (blue). GFP-Itch is shown in green. Cells were visualized using DIC.

    Techniques Used: Transfection

    LITAF and Itch cellular localization. BGMK cells were transiently transfected for 8 or 24 hours with (A) FLAG-LITAF or (B) GFP-Itch. Live cells were initially incubated with LysoTracker followed by fixation and permeabilization. Cells then underwent indirect immunofluorescence using anti-IGF-IIR (trans Golgi-network; blue) and/or anti-FLAG antibodies (LITAF; red). GFP-Itch is shown in green. Differential interference contrast (DIC) was used to visualize cells and images were captured using a laser scanning confocal microscope.
    Figure Legend Snippet: LITAF and Itch cellular localization. BGMK cells were transiently transfected for 8 or 24 hours with (A) FLAG-LITAF or (B) GFP-Itch. Live cells were initially incubated with LysoTracker followed by fixation and permeabilization. Cells then underwent indirect immunofluorescence using anti-IGF-IIR (trans Golgi-network; blue) and/or anti-FLAG antibodies (LITAF; red). GFP-Itch is shown in green. Differential interference contrast (DIC) was used to visualize cells and images were captured using a laser scanning confocal microscope.

    Techniques Used: Transfection, Incubation, Immunofluorescence, Microscopy

    Mutation of both PPXY domains disrupts Itch and LITAF interaction. (A) Extracts from HEK-293T cells transfected with GFP-LITAF WT or GFP-LITAF Y23,61A were incubated with either GST alone, GST-Itch WT, GST-Itch PRD or GST-Itch WW pre-coupled to glutathione-Sepharose. Aliquot from total cell lysate (CL) and proteins specifically bound to the beads were processed by immunoblot with a polyclonal antibody against GFP. (B) HEK-293T cells were transiently transfected with either GFP-LITAF WT, GFP-LITAF Y61A, GFP-LITAF Y23A or GFP-LITAF Y23,61A. Aliquots of CL were processed by immunoblot with GFP antibody to show protein expression. The rest of the extracts were incubated with either GST or GST-Itch WW fusion proteins pre-coupled to gluthatione-Sepharose. Proteins specifically bound to the beads were immunoblotted with GFP antibody to reveal protein interactions. The bands representing the GST-fusions in the Ponceau staining are marked by a red asterisk. Additional staining in the GST-Itch-WT lane likely represents degradation products of the fusion protein. (C) 293T cells were co-transfected with constant amount of rLuc-Itch and various amounts of either GFP-LITAF Y61A or GFP-LITAF Y23,61A. The graph is a representative example of the saturation studies performed to provide evidence for a specific interaction between the proteins. BRET ratios were plotted as a function of the excited GFP activity to total rLuc activity ratio, allowing comparison of BRET ratios between GFP-LITAF Y61A and GFP-LITAF Y23,61A when expressed at the same level. (D) Quantification of the interaction between Itch WW domains and the different LITAF constructs. The densitometry of GFP signal in the fraction bound to GST-Itch-WW beads relative to the densitometry of the GFP signal in 1/10 volume of protein extract is represented as described in materials and methods . Data are mean ± s.e.m. from n = 4 experiments. * p
    Figure Legend Snippet: Mutation of both PPXY domains disrupts Itch and LITAF interaction. (A) Extracts from HEK-293T cells transfected with GFP-LITAF WT or GFP-LITAF Y23,61A were incubated with either GST alone, GST-Itch WT, GST-Itch PRD or GST-Itch WW pre-coupled to glutathione-Sepharose. Aliquot from total cell lysate (CL) and proteins specifically bound to the beads were processed by immunoblot with a polyclonal antibody against GFP. (B) HEK-293T cells were transiently transfected with either GFP-LITAF WT, GFP-LITAF Y61A, GFP-LITAF Y23A or GFP-LITAF Y23,61A. Aliquots of CL were processed by immunoblot with GFP antibody to show protein expression. The rest of the extracts were incubated with either GST or GST-Itch WW fusion proteins pre-coupled to gluthatione-Sepharose. Proteins specifically bound to the beads were immunoblotted with GFP antibody to reveal protein interactions. The bands representing the GST-fusions in the Ponceau staining are marked by a red asterisk. Additional staining in the GST-Itch-WT lane likely represents degradation products of the fusion protein. (C) 293T cells were co-transfected with constant amount of rLuc-Itch and various amounts of either GFP-LITAF Y61A or GFP-LITAF Y23,61A. The graph is a representative example of the saturation studies performed to provide evidence for a specific interaction between the proteins. BRET ratios were plotted as a function of the excited GFP activity to total rLuc activity ratio, allowing comparison of BRET ratios between GFP-LITAF Y61A and GFP-LITAF Y23,61A when expressed at the same level. (D) Quantification of the interaction between Itch WW domains and the different LITAF constructs. The densitometry of GFP signal in the fraction bound to GST-Itch-WW beads relative to the densitometry of the GFP signal in 1/10 volume of protein extract is represented as described in materials and methods . Data are mean ± s.e.m. from n = 4 experiments. * p

    Techniques Used: Mutagenesis, Transfection, Incubation, Expressing, Staining, Bioluminescence Resonance Energy Transfer, Activity Assay, Construct

    LITAF interacts with Itch in vitro and in vivo . (A) HEK-293T cells were transiently transfected with FLAG-Itch with or without co-transfection of myc-LITAF. Total cell lysates were blotted with anti-FLAG and anti-myc to show protein expression (lower panel), and immunoprecipitated with anti-FLAG to reveal LITAF co-immunoprecipitation (upper panel). (B) Extracts from 293T cells transfected with myc-LITAF were incubated with either GST alone, GST-Itch WT, GST-Itch PRD or GST-Itch WW pre-coupled to glutathione-Sepharose. Input proteins is shown in the first lane (CL). Proteins bound to GST beads are shown in the next lanes. Immunoblotting with anti- myc antibodies shows the presence of myc-LITAF (upper panel). Total gel loading is shown by ponceau staining of the blot to reveal GST loading. The bands representing the GST-fusions in the Ponceau staining are marked by a red asterisk. Additional staining in the GST-Itch-WT lane likely represents degradation products of the fusion protein. (C) 293T cells were co-transfected with constant amount of rLuc-Itch and various amounts of either GFP alone or GFP-LITAF. The graph is a representative example of the saturation studies performed to provide evidence for a specific interaction between the proteins. BRET ratios were plotted as a function of the excited GFP activity to total rLuc activity ratio, allowing comparison of BRET ratios between the negative control GFP and GFP-LITAF when expressed at the same level.
    Figure Legend Snippet: LITAF interacts with Itch in vitro and in vivo . (A) HEK-293T cells were transiently transfected with FLAG-Itch with or without co-transfection of myc-LITAF. Total cell lysates were blotted with anti-FLAG and anti-myc to show protein expression (lower panel), and immunoprecipitated with anti-FLAG to reveal LITAF co-immunoprecipitation (upper panel). (B) Extracts from 293T cells transfected with myc-LITAF were incubated with either GST alone, GST-Itch WT, GST-Itch PRD or GST-Itch WW pre-coupled to glutathione-Sepharose. Input proteins is shown in the first lane (CL). Proteins bound to GST beads are shown in the next lanes. Immunoblotting with anti- myc antibodies shows the presence of myc-LITAF (upper panel). Total gel loading is shown by ponceau staining of the blot to reveal GST loading. The bands representing the GST-fusions in the Ponceau staining are marked by a red asterisk. Additional staining in the GST-Itch-WT lane likely represents degradation products of the fusion protein. (C) 293T cells were co-transfected with constant amount of rLuc-Itch and various amounts of either GFP alone or GFP-LITAF. The graph is a representative example of the saturation studies performed to provide evidence for a specific interaction between the proteins. BRET ratios were plotted as a function of the excited GFP activity to total rLuc activity ratio, allowing comparison of BRET ratios between the negative control GFP and GFP-LITAF when expressed at the same level.

    Techniques Used: In Vitro, In Vivo, Transfection, Cotransfection, Expressing, Immunoprecipitation, Incubation, Staining, Bioluminescence Resonance Energy Transfer, Activity Assay, Negative Control

    Mutation of both PPXY domains disrupts Itch and LITAF co-localization. (A) Site-directed mutagenesis was used to mutate the two PPXY motifs (either individually or together) located in the N-terminus of GFP-LITAF. (B) Expression constructs containing each mutated GFP-LITAF construct and FLAG-Itch were co-transfected into BGMK cells and 16 hours post-transfection, cells were incubated with LysoTracker (blue), fixed, and processed by indirect immunofluorescence to detect Itch (anti-FLAG; green), LITAF (red), and the trans-Golgi network (anti-IGF-IIR; blue). Cells were visualized using DIC.
    Figure Legend Snippet: Mutation of both PPXY domains disrupts Itch and LITAF co-localization. (A) Site-directed mutagenesis was used to mutate the two PPXY motifs (either individually or together) located in the N-terminus of GFP-LITAF. (B) Expression constructs containing each mutated GFP-LITAF construct and FLAG-Itch were co-transfected into BGMK cells and 16 hours post-transfection, cells were incubated with LysoTracker (blue), fixed, and processed by indirect immunofluorescence to detect Itch (anti-FLAG; green), LITAF (red), and the trans-Golgi network (anti-IGF-IIR; blue). Cells were visualized using DIC.

    Techniques Used: Mutagenesis, Expressing, Construct, Transfection, Incubation, Immunofluorescence

    4) Product Images from "Pathophysiological Mechanisms of Autosomal Dominant Congenital Stromal Corneal Dystrophy"

    Article Title: Pathophysiological Mechanisms of Autosomal Dominant Congenital Stromal Corneal Dystrophy

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2011.07.026

    952delT Dcn transgenic mice express both wild-type and mutant decorin. A: Two independent families with human CSCD have been reported. A single base pair deletion in the coding sequence at either 941 (delC) or 967 (delT) caused frameshift mutations and a truncation of the C-terminal 33 amino acids. In mice, a delG at 926 or a delT at 952 (highlighted in red) will result in a comparable frameshift mutation. The new stop codon TGA (highlighted in red) will yield a C-terminal truncated decorin comparable to that in human CSCD. B: A Cre-on approach was used to create a transgenic mouse carrying a mutant decorin cDNA with a deleted T at 952. A stop codon flanked with LoxP ( red triangle ) elements was inserted between a ubiquitous CAG promoter and the mutant decorin cDNA. Corneal stromal targeting of mutant decorin was generated through breeding with Kera -cre mouse. The mouse model was bred into different backgrounds, including decorin wild-type, heterozygous, and deficient backgrounds. C: Immunoblot analyses show that the transgenic mice in a decorin wild-type background expresses two decorin bands after sequential Chondroitinase ABC and PNGase F digestion, one migrating with the wild-type decorin core, the other migrating faster at ∼37 kDa, consistent with the C-terminal truncation. The level of mutant decorin was substantially less than the wild-type decorin. These qualitative immunoblots were overloaded/exposed to show the presence or absence of the truncated decorin core in mutant and wild-type corneas, respectively. The antibody used was generated against the N-terminal 17 amino acids of the decorin protein core and thus recognizes both the native and the C-terminal–truncated species. D: Breeding the mutant into a decorin-null background allowed the localization of the mutant protein core. Mutant decorin expression was identified by immunolocalization in a 952delT Dcn mouse in decorin-deficient background ( yellow arrow indicates the positive reactivity of mutant decorin in the corneal stroma). Scale bar = 25 μm.
    Figure Legend Snippet: 952delT Dcn transgenic mice express both wild-type and mutant decorin. A: Two independent families with human CSCD have been reported. A single base pair deletion in the coding sequence at either 941 (delC) or 967 (delT) caused frameshift mutations and a truncation of the C-terminal 33 amino acids. In mice, a delG at 926 or a delT at 952 (highlighted in red) will result in a comparable frameshift mutation. The new stop codon TGA (highlighted in red) will yield a C-terminal truncated decorin comparable to that in human CSCD. B: A Cre-on approach was used to create a transgenic mouse carrying a mutant decorin cDNA with a deleted T at 952. A stop codon flanked with LoxP ( red triangle ) elements was inserted between a ubiquitous CAG promoter and the mutant decorin cDNA. Corneal stromal targeting of mutant decorin was generated through breeding with Kera -cre mouse. The mouse model was bred into different backgrounds, including decorin wild-type, heterozygous, and deficient backgrounds. C: Immunoblot analyses show that the transgenic mice in a decorin wild-type background expresses two decorin bands after sequential Chondroitinase ABC and PNGase F digestion, one migrating with the wild-type decorin core, the other migrating faster at ∼37 kDa, consistent with the C-terminal truncation. The level of mutant decorin was substantially less than the wild-type decorin. These qualitative immunoblots were overloaded/exposed to show the presence or absence of the truncated decorin core in mutant and wild-type corneas, respectively. The antibody used was generated against the N-terminal 17 amino acids of the decorin protein core and thus recognizes both the native and the C-terminal–truncated species. D: Breeding the mutant into a decorin-null background allowed the localization of the mutant protein core. Mutant decorin expression was identified by immunolocalization in a 952delT Dcn mouse in decorin-deficient background ( yellow arrow indicates the positive reactivity of mutant decorin in the corneal stroma). Scale bar = 25 μm.

    Techniques Used: Transgenic Assay, Mouse Assay, Mutagenesis, Sequencing, Generated, Western Blot, Expressing

    5) Product Images from "GHK Peptide Inhibits Bleomycin-Induced Pulmonary Fibrosis in Mice by Suppressing TGFβ1/Smad-Mediated Epithelial-to-Mesenchymal Transition"

    Article Title: GHK Peptide Inhibits Bleomycin-Induced Pulmonary Fibrosis in Mice by Suppressing TGFβ1/Smad-Mediated Epithelial-to-Mesenchymal Transition

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00904

    Effects of GHK on TGF-β1 mRNA and protein expression in mice with BLM-induced fibrosis. (A) TGF-β1 expression levels in lung tissues in each group were determined by real-time PCR. (B) TGF-β1 activity in lung tissues was measured by an ELISA kit. (C) TGF-β1 protein expression in lung tissues in each group was measured by western blot analysis. (D) The densitometry values of the proteins were normalized to those of β-actin. All data represent the mean ± SEM of three independent experiments performed in triplicate. Statistical analysis was performed by one-way ANOVA and Turkey’s multiple-comparison test; compared with control group, ## P
    Figure Legend Snippet: Effects of GHK on TGF-β1 mRNA and protein expression in mice with BLM-induced fibrosis. (A) TGF-β1 expression levels in lung tissues in each group were determined by real-time PCR. (B) TGF-β1 activity in lung tissues was measured by an ELISA kit. (C) TGF-β1 protein expression in lung tissues in each group was measured by western blot analysis. (D) The densitometry values of the proteins were normalized to those of β-actin. All data represent the mean ± SEM of three independent experiments performed in triplicate. Statistical analysis was performed by one-way ANOVA and Turkey’s multiple-comparison test; compared with control group, ## P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

    6) Product Images from "Therapeutic relevance of the PP2A-B55 inhibitory kinase MASTL/Greatwall in breast cancer"

    Article Title: Therapeutic relevance of the PP2A-B55 inhibitory kinase MASTL/Greatwall in breast cancer

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-017-0024-0

    Breast cancer cells require MASTL kinase activity a Immunoblotting analysis of MASTL protein level in iCas9 and isg MASTL cells expressing murine wild-type (WT) or kinase dead (G43S) Mastl forms fused to GFP in the presence and absence of doxycycline (4 days treatment). b FACS profiles showing accumulation of ≥4n cells after depletion of MASTL and rescue by the WT, but not G43S, murine Mastl forms. The graph shows the fold induction in the percentage of ≥4n cells upon doxycycline (Dox) treatment. Bars indicate means + SD for three independent experiments. c Mastl WT, but not G43S, rescues cell growth in colony formation assays. Graph on the left panel shows cell growth quantification, where cell proliferation in the absence of doxycycline was set as 100%. Bars indicate means + SD for three independent experiments. The middle and right panels show the crystal violet staining of one representative experiment. ***, p
    Figure Legend Snippet: Breast cancer cells require MASTL kinase activity a Immunoblotting analysis of MASTL protein level in iCas9 and isg MASTL cells expressing murine wild-type (WT) or kinase dead (G43S) Mastl forms fused to GFP in the presence and absence of doxycycline (4 days treatment). b FACS profiles showing accumulation of ≥4n cells after depletion of MASTL and rescue by the WT, but not G43S, murine Mastl forms. The graph shows the fold induction in the percentage of ≥4n cells upon doxycycline (Dox) treatment. Bars indicate means + SD for three independent experiments. c Mastl WT, but not G43S, rescues cell growth in colony formation assays. Graph on the left panel shows cell growth quantification, where cell proliferation in the absence of doxycycline was set as 100%. Bars indicate means + SD for three independent experiments. The middle and right panels show the crystal violet staining of one representative experiment. ***, p

    Techniques Used: Activity Assay, Expressing, FACS, Staining

    7) Product Images from "Novel human and mouse genes encoding an acid phosphatase family member and its downregulation in W/WV mouse jejunum"

    Article Title: Novel human and mouse genes encoding an acid phosphatase family member and its downregulation in W/WV mouse jejunum

    Journal: Gut

    doi:

    Expression of acid phosphatase-like protein 1 (ACPL1) using reverse transcription-polymerase chain reaction analysis in jejunum tissues from two wild-type and two W/W V mutant mice maintained under fed and starved conditions. F and S indicate samples of fed and staved mice, respectively. Glyceraldehyde-3- phosphate dehydrogenase (G3PDH) levels were measured as a control. Expression of the 54M clone was considerably reduced in the jejunums of W/W V mice compared with age matched wild-type mice.
    Figure Legend Snippet: Expression of acid phosphatase-like protein 1 (ACPL1) using reverse transcription-polymerase chain reaction analysis in jejunum tissues from two wild-type and two W/W V mutant mice maintained under fed and starved conditions. F and S indicate samples of fed and staved mice, respectively. Glyceraldehyde-3- phosphate dehydrogenase (G3PDH) levels were measured as a control. Expression of the 54M clone was considerably reduced in the jejunums of W/W V mice compared with age matched wild-type mice.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Mouse Assay

    8) Product Images from "Otogelin: A glycoprotein specific to the acellular membranes of the inner ear"

    Article Title: Otogelin: A glycoprotein specific to the acellular membranes of the inner ear

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

    doi:

    ( A ) Deduced amino acid sequence of otogelin. The signal peptide is underlined. Arrows delineate the different protein domains. WF D-type domains D1 (residues 135–498), D2 (500–870), D′ (873–971, truncated domain), D3 (972–1450), and D4 (2093–2459) share between each other and with the D domains of WF a sequence similarity of about 50%. The D3 domain contains a unique 103-residue insertion (position 1242–1394), which is shaded. The conserved residues of the multimerization site are shaded. Five WF B-type domains, B1 (2460–2492), B2 (2497–2527), B3 (2532–2563), B4 (2266–2597), B5 (2602–2632) are shown. The TSP domain (1451–2036) contains 13% threonines, 13% serines, and 15% prolines and is devoid of cysteine; CT, carboxyl-terminal end (2825–2910). The potential N- glycosylation sites are underlined and in bold. | indicates the additional 3′ RACE PCR alternatively spliced sequence presented at the end of the sequence. The cysteine involved in the dimerization of WF and TGF-β2 (position 2873) and the conserved glycine (position 2852) are indicated by an asterisk. The cysteines involved in the TGF-β2 knot cystine structure formation are numbered. ( B ) Schematic representation of the structure of otogelin. The thick bar indicates the predicted signal peptide.
    Figure Legend Snippet: ( A ) Deduced amino acid sequence of otogelin. The signal peptide is underlined. Arrows delineate the different protein domains. WF D-type domains D1 (residues 135–498), D2 (500–870), D′ (873–971, truncated domain), D3 (972–1450), and D4 (2093–2459) share between each other and with the D domains of WF a sequence similarity of about 50%. The D3 domain contains a unique 103-residue insertion (position 1242–1394), which is shaded. The conserved residues of the multimerization site are shaded. Five WF B-type domains, B1 (2460–2492), B2 (2497–2527), B3 (2532–2563), B4 (2266–2597), B5 (2602–2632) are shown. The TSP domain (1451–2036) contains 13% threonines, 13% serines, and 15% prolines and is devoid of cysteine; CT, carboxyl-terminal end (2825–2910). The potential N- glycosylation sites are underlined and in bold. | indicates the additional 3′ RACE PCR alternatively spliced sequence presented at the end of the sequence. The cysteine involved in the dimerization of WF and TGF-β2 (position 2873) and the conserved glycine (position 2852) are indicated by an asterisk. The cysteines involved in the TGF-β2 knot cystine structure formation are numbered. ( B ) Schematic representation of the structure of otogelin. The thick bar indicates the predicted signal peptide.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    9) Product Images from "Cloning and expression analysis of prohibitin mRNA in canine mammary tumors"

    Article Title: Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

    Journal: The Journal of Veterinary Medical Science

    doi: 10.1292/jvms.14-0239

    Sequence of canine prohibitin cDNA compared with the human sequence. Asterisks show corresponding nucleotides; “F” and “R” indicate the annealing sites of the prohibitin-specific primer set; and “1” and “2” show the initiation and termination codons, respectively.
    Figure Legend Snippet: Sequence of canine prohibitin cDNA compared with the human sequence. Asterisks show corresponding nucleotides; “F” and “R” indicate the annealing sites of the prohibitin-specific primer set; and “1” and “2” show the initiation and termination codons, respectively.

    Techniques Used: Sequencing

    10) Product Images from "Biodistribution and blood clearance of plasmid DNA administered in arginine peptide complexes"

    Article Title: Biodistribution and blood clearance of plasmid DNA administered in arginine peptide complexes

    Journal: Genetic Vaccines and Therapy

    doi: 10.1186/1479-0556-9-13

    mRNA expression levels of the target gene in various organs . mRNA levels were evaluated using real time RT-PCR. Plasmid DNA (100 μg) complexed with arginine peptide at an N/P ratio of 3:1 was intraperitoneally administered to mice. Mice were sacrificed at the indicated time points, and total RNA was extracted from the organs. After preparation of cDNA, PCR amplification of luciferase and GAPDH genes was performed using the specific primers described in the Materials and Methods section. Results are expressed as means ± S.D. for at least 3 different experiments.
    Figure Legend Snippet: mRNA expression levels of the target gene in various organs . mRNA levels were evaluated using real time RT-PCR. Plasmid DNA (100 μg) complexed with arginine peptide at an N/P ratio of 3:1 was intraperitoneally administered to mice. Mice were sacrificed at the indicated time points, and total RNA was extracted from the organs. After preparation of cDNA, PCR amplification of luciferase and GAPDH genes was performed using the specific primers described in the Materials and Methods section. Results are expressed as means ± S.D. for at least 3 different experiments.

    Techniques Used: Expressing, Quantitative RT-PCR, Plasmid Preparation, Mouse Assay, Polymerase Chain Reaction, Amplification, Luciferase

    Effects of DNA dose on plasmid DNA expression after delivery in arginine/DNA complexes . Various amounts of plasmid DNA complexed with arginine peptide at an N/P ratio of 3:1 were intraperitoneally administered to mice, and mRNA levels were evaluated using real time RT-PCR. Total RNA was extracted from the organs. After preparation of cDNA, PCR amplification of luciferase and GAPDH genes was performed using the specific primers described in the Materials and Methods section. Results are expressed as means ± S.D. for at least 3 different experiments.
    Figure Legend Snippet: Effects of DNA dose on plasmid DNA expression after delivery in arginine/DNA complexes . Various amounts of plasmid DNA complexed with arginine peptide at an N/P ratio of 3:1 were intraperitoneally administered to mice, and mRNA levels were evaluated using real time RT-PCR. Total RNA was extracted from the organs. After preparation of cDNA, PCR amplification of luciferase and GAPDH genes was performed using the specific primers described in the Materials and Methods section. Results are expressed as means ± S.D. for at least 3 different experiments.

    Techniques Used: Plasmid Preparation, Expressing, Mouse Assay, Quantitative RT-PCR, Polymerase Chain Reaction, Amplification, Luciferase

    11) Product Images from "Membrane Fusion by VAMP3 and Plasma Membrane t-SNAREs"

    Article Title: Membrane Fusion by VAMP3 and Plasma Membrane t-SNAREs

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2007.06.008

    Expression of flipped SNARE proteins. (A) Twenty-four hours after transfection, whole-cell lysates of the COS-7 cells transfected with flipped VAMP3 or empty pcDNA3.1(+) vector (Mock) were immunoblotted with an antibody to Myc tag. (B) Whole cell lysates of COS-7 cells cotransfected with flipped SNAP-23 and flipped syntaxin4, flipped syntaxin4(Δ111) or flipped syntaxin4 H3 were immunoblotted with an antibody to Myc tag to detect the syntaxin4 proteins. Treatment with tunicamycin (5 μg/ml, overnight) did not change the mobility of flipped syntaxin4 proteins. (C) Whole cell lysates of COS-7 cells transfected with flipped syntaxin4 and flipped SNAP-23(N) or flipped SNAP-23 were immunoblotted with an antibody to FLAG tag to detect the SNAP-23 proteins. Flipped SNAP-23(N) is the SNAP-23 construct that contains the putative N-glycosylation sites. In flipped SNAP-23, the N-glycosylation sites have been removed by point mutations. Treatment with tunicamycin (5 μg/ml, overnight) increased the mobility of flipped SNAP-23(N) proteins to that of flipped SNAP-23 proteins.
    Figure Legend Snippet: Expression of flipped SNARE proteins. (A) Twenty-four hours after transfection, whole-cell lysates of the COS-7 cells transfected with flipped VAMP3 or empty pcDNA3.1(+) vector (Mock) were immunoblotted with an antibody to Myc tag. (B) Whole cell lysates of COS-7 cells cotransfected with flipped SNAP-23 and flipped syntaxin4, flipped syntaxin4(Δ111) or flipped syntaxin4 H3 were immunoblotted with an antibody to Myc tag to detect the syntaxin4 proteins. Treatment with tunicamycin (5 μg/ml, overnight) did not change the mobility of flipped syntaxin4 proteins. (C) Whole cell lysates of COS-7 cells transfected with flipped syntaxin4 and flipped SNAP-23(N) or flipped SNAP-23 were immunoblotted with an antibody to FLAG tag to detect the SNAP-23 proteins. Flipped SNAP-23(N) is the SNAP-23 construct that contains the putative N-glycosylation sites. In flipped SNAP-23, the N-glycosylation sites have been removed by point mutations. Treatment with tunicamycin (5 μg/ml, overnight) increased the mobility of flipped SNAP-23(N) proteins to that of flipped SNAP-23 proteins.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, FLAG-tag, Construct

    Expression of flipped SNARE proteins on the cell surface. (A) Twenty-four hours after transfection, unpermeabilized COS-7 cells transfected with flipped VAMP3 were stained with an antibody to Myc tag to detect cell surface distribution of VAMP3. (B) COS-7 cells were mock transfected (no DNA), co-transfected with flipped syntaxin4 and flipped SNAP-23, or cotransfected with flipped syntaxin1 H3 and flipped SNAP-25. Twenty-four hours after transfection, unpermeabilized COS-7 cells were dual labeled with antibodies. Syntaxin4 and syntaxin1 H3 proteins were stained with an antibody to Myc tag (green), and SNAP-23 proteins were detected with an antibody to FLAG tag (red). SNAP-25 proteins on the cell surface were detected with an antibody to SNAP-25 (red). Confocal images were collected using the same settings. Representative images of three experiments are shown. Scale bar, 20 μm.
    Figure Legend Snippet: Expression of flipped SNARE proteins on the cell surface. (A) Twenty-four hours after transfection, unpermeabilized COS-7 cells transfected with flipped VAMP3 were stained with an antibody to Myc tag to detect cell surface distribution of VAMP3. (B) COS-7 cells were mock transfected (no DNA), co-transfected with flipped syntaxin4 and flipped SNAP-23, or cotransfected with flipped syntaxin1 H3 and flipped SNAP-25. Twenty-four hours after transfection, unpermeabilized COS-7 cells were dual labeled with antibodies. Syntaxin4 and syntaxin1 H3 proteins were stained with an antibody to Myc tag (green), and SNAP-23 proteins were detected with an antibody to FLAG tag (red). SNAP-25 proteins on the cell surface were detected with an antibody to SNAP-25 (red). Confocal images were collected using the same settings. Representative images of three experiments are shown. Scale bar, 20 μm.

    Techniques Used: Expressing, Transfection, Staining, Labeling, FLAG-tag

    12) Product Images from "Characterization of a Plasmodium berghei sexual stage antigen PbPH as a new candidate for malaria transmission-blocking vaccine"

    Article Title: Characterization of a Plasmodium berghei sexual stage antigen PbPH as a new candidate for malaria transmission-blocking vaccine

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-016-1459-8

    Functional analysis of PbPH during parasite development. a Mice were infected with P. berghei or Δpbph parasite and parasitemia was monitored for 11 days. b Gametocytemias in mice infected with wild-type (WT) and Δpbph parasites. ** P
    Figure Legend Snippet: Functional analysis of PbPH during parasite development. a Mice were infected with P. berghei or Δpbph parasite and parasitemia was monitored for 11 days. b Gametocytemias in mice infected with wild-type (WT) and Δpbph parasites. ** P

    Techniques Used: Functional Assay, Mouse Assay, Infection

    Bioinformatic analyses of PbPH. a Alignment of protein sequences of PH domain proteins in Plasmodium species: P. berghei ( Pb ), P. falciparum ( Pf ), P. yoelii ( Py ), P. chabaudi ( Pc ), P. vivax ( Pv ), and P. knowlesi ( Pk ). Amino acids conserved across six species are marked: identical (black), similar (gray). The PH domain is highlighted. b Schematic diagram of PbPH showing the locations of a signal peptide (red) and a PH domain (blue). The E. coli expression segment shows amino acids 21 to 286
    Figure Legend Snippet: Bioinformatic analyses of PbPH. a Alignment of protein sequences of PH domain proteins in Plasmodium species: P. berghei ( Pb ), P. falciparum ( Pf ), P. yoelii ( Py ), P. chabaudi ( Pc ), P. vivax ( Pv ), and P. knowlesi ( Pk ). Amino acids conserved across six species are marked: identical (black), similar (gray). The PH domain is highlighted. b Schematic diagram of PbPH showing the locations of a signal peptide (red) and a PH domain (blue). The E. coli expression segment shows amino acids 21 to 286

    Techniques Used: Expressing

    Knockout of pbph gene from P. berghei parasite. a A schematic shows the wild type pbph locus, transfection construct, and the recombined locus. Through a double-crossover strategy the pbph locus was replaced with the dhfr -expressing cassette. Primers 1–6 used to detect gene knockout are marked. b PCR detection of wild-type and Δpbph parasite genomes. Lane 1: primers 1 + 2 (910 bp); Lane 2: primer 1 + 3 (990 bp); and Lane 3: primer 5 + 6 (1, 040 bp)
    Figure Legend Snippet: Knockout of pbph gene from P. berghei parasite. a A schematic shows the wild type pbph locus, transfection construct, and the recombined locus. Through a double-crossover strategy the pbph locus was replaced with the dhfr -expressing cassette. Primers 1–6 used to detect gene knockout are marked. b PCR detection of wild-type and Δpbph parasite genomes. Lane 1: primers 1 + 2 (910 bp); Lane 2: primer 1 + 3 (990 bp); and Lane 3: primer 5 + 6 (1, 040 bp)

    Techniques Used: Knock-Out, Transfection, Construct, Expressing, Gene Knockout, Polymerase Chain Reaction

    Expression and localization of PbPH in the parasites. a Western blot of PbPH in asexual- and sexual-stage parasites. Parasite antigens of schizonts, gametocytes, and ookinetes (10 μg/lane) were incubated with anti-PbPH sera (1:200) from immunized mice. Protein loading is estimated using anti-Hsp70 serum (1:200). b IFA analysis. Blood smears containing parasite samples were stained at different times after collection of P. berghei -infected blood. Parasites were fixed and stained with anti-PbPH sera (1:200) from recombinant PbPH-immunized mice and then with FITC-conjugated goat anti-mouse IgG (green). Nuclei were stained by DAPI (blue). Pbs21 mAb clone 13.1 serves as a positive control. Scale - bar: 5 μm
    Figure Legend Snippet: Expression and localization of PbPH in the parasites. a Western blot of PbPH in asexual- and sexual-stage parasites. Parasite antigens of schizonts, gametocytes, and ookinetes (10 μg/lane) were incubated with anti-PbPH sera (1:200) from immunized mice. Protein loading is estimated using anti-Hsp70 serum (1:200). b IFA analysis. Blood smears containing parasite samples were stained at different times after collection of P. berghei -infected blood. Parasites were fixed and stained with anti-PbPH sera (1:200) from recombinant PbPH-immunized mice and then with FITC-conjugated goat anti-mouse IgG (green). Nuclei were stained by DAPI (blue). Pbs21 mAb clone 13.1 serves as a positive control. Scale - bar: 5 μm

    Techniques Used: Expressing, Western Blot, Incubation, Mouse Assay, Immunofluorescence, Staining, Infection, Recombinant, Positive Control

    13) Product Images from "Protein Kinase G Dynamically Modulates TASK1-Mediated Leak K+ Currents in Cholinergic Neurons of the Basal Forebrain"

    Article Title: Protein Kinase G Dynamically Modulates TASK1-Mediated Leak K+ Currents in Cholinergic Neurons of the Basal Forebrain

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.5407-09.2010

    Effects of 8-Br-cGMP and KT5823 on TASK1 mutants H98N and K210N. A , Voltage command pulse ( a ). Superimposed traces of current responses obtained at pH 6.3, 7.3, and 8.3 in H98N channels in the absence (black traces) and presence (red traces) of 8-Br-cGMP ( b ) or KT5823 ( c ). Sample current traces at pH 8.3 were obtained from different HEK cells in b and c . Calibration bars for time scale and current amplitude shown in b also apply to all other current responses in A and B . B , Superimposed traces of current responses obtained at pH 6.3, 7.3, and 8.3 in K210N channels in the absence (black traces) and presence (red traces) of 8-Br-cGMP ( a ) or KT5823 ( b ). C , Pooled data showing the mean conductance normalized to that at pH 8.3 in the current responses obtained at pH 6.3, at pH 7.3 and at pH 8.3 in WT TASK1 channels (open columns, n = 7), K210N channels (black columns, n = 6) and H98N channels (gray columns, n = 5). * p
    Figure Legend Snippet: Effects of 8-Br-cGMP and KT5823 on TASK1 mutants H98N and K210N. A , Voltage command pulse ( a ). Superimposed traces of current responses obtained at pH 6.3, 7.3, and 8.3 in H98N channels in the absence (black traces) and presence (red traces) of 8-Br-cGMP ( b ) or KT5823 ( c ). Sample current traces at pH 8.3 were obtained from different HEK cells in b and c . Calibration bars for time scale and current amplitude shown in b also apply to all other current responses in A and B . B , Superimposed traces of current responses obtained at pH 6.3, 7.3, and 8.3 in K210N channels in the absence (black traces) and presence (red traces) of 8-Br-cGMP ( a ) or KT5823 ( b ). C , Pooled data showing the mean conductance normalized to that at pH 8.3 in the current responses obtained at pH 6.3, at pH 7.3 and at pH 8.3 in WT TASK1 channels (open columns, n = 7), K210N channels (black columns, n = 6) and H98N channels (gray columns, n = 5). * p

    Techniques Used:

    Expression of TASK1, TASK3, and ChAT in MS/DB neurons. A , A type II neuron displaying either a late spiking due to A-like K + current in response to a depolarizing current pulse applied at −93 mV (right) or a regular spiking at −70 mV (left). B , mRNA expression profile of TASK1, TASK3, and ChAT obtained from the type II neuron shown in A , revealed by single-cell RT-PCR. Note that TASK1 mRNA was expressed together with ChAT mRNA in the type II neuron. C , mRNA expression profile of TASK1 and TASK3 obtained from a TMN neuron. The amplified PCR fragments were consistent with the respective lengths predicted by the two subunit primers. D , E , Confocal photomicrographs showing immunoreactivity for ChAT (green) and TASK1 or TASK3 (red) in neurons of MS/DB nuclei. As revealed in the merged images, both cholinergic neurons (filled arrowheads) and noncholinergic neurons (open arrowheads) were immunopositive for TASK1 antibody ( D ), while neither cholinergic (filled arrowheads) nor noncholinergic neurons were immunopositive for TASK3 antibody ( E ).
    Figure Legend Snippet: Expression of TASK1, TASK3, and ChAT in MS/DB neurons. A , A type II neuron displaying either a late spiking due to A-like K + current in response to a depolarizing current pulse applied at −93 mV (right) or a regular spiking at −70 mV (left). B , mRNA expression profile of TASK1, TASK3, and ChAT obtained from the type II neuron shown in A , revealed by single-cell RT-PCR. Note that TASK1 mRNA was expressed together with ChAT mRNA in the type II neuron. C , mRNA expression profile of TASK1 and TASK3 obtained from a TMN neuron. The amplified PCR fragments were consistent with the respective lengths predicted by the two subunit primers. D , E , Confocal photomicrographs showing immunoreactivity for ChAT (green) and TASK1 or TASK3 (red) in neurons of MS/DB nuclei. As revealed in the merged images, both cholinergic neurons (filled arrowheads) and noncholinergic neurons (open arrowheads) were immunopositive for TASK1 antibody ( D ), while neither cholinergic (filled arrowheads) nor noncholinergic neurons were immunopositive for TASK3 antibody ( E ).

    Techniques Used: Expressing, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    Modulation of TASK1 currents by basal level of PKG. A , Photomicrographs of fluorescence images showing immunoreactivity for TASK1 channels (top) and bright-field images showing the presence of HEK cells in each experiment (bottom). The HEK cells transfected with TASK1 displaying unambiguous immunoreactivity along the plasma membrane and partly in the cytoplasm ( a ). The HEK cells transfected with mock displaying no intense immunoreactivity more than diffuse background fluorescence ( b ). The HEK cells transfected with TASK1 displaying no intense immunoreactivity in the absence of the primary antibody against TASK1 ( c ). Ba , Top, Voltage command pulse. Bottom, Superimposed traces of small inwardly rectifying current responses obtained at pH 6.3, 7.3, and 8.3 in the PKG-unloaded and PKGII-loaded mock-transfected HEK cells. Note no apparent pH-sensitive K + current. Bb , In the PKG-unloaded (open columns, n = 5) and PKGII-loaded (gray columns, n = 8) mock-transfected HEK cells, the mean conductances normalized to those at pH 6.3 in the current responses obtained at pH 6.3 (1.0 and 1.0, respectively), at pH 7.3 (1.2 ± 0.3 and 1.2 ± 0.2, respectively), and at pH 8.3 (1.3 ± 0.4 and 1.2 ± 0.2, respectively). C , Top, Voltage command pulses. Bottom, Sample current traces obtained at pH 6.3, 7.3, and 8.3 in TASK1-transfected HEK cells in the absence ( a ) and presence ( b ) of PKGII. Black dotted curves obtained by fitting the TASK1 currents at pH 8.3 (red traces) with GHK equation. D , Pooled data showing the mean values of normalized conductances ( a ) and the mean values of scaled conductances ( b ) at pH 6.3, 7.3, and 8.3 in the absence (open columns, n = 5) and presence (gray columns, n = 6) of PKGII. * p
    Figure Legend Snippet: Modulation of TASK1 currents by basal level of PKG. A , Photomicrographs of fluorescence images showing immunoreactivity for TASK1 channels (top) and bright-field images showing the presence of HEK cells in each experiment (bottom). The HEK cells transfected with TASK1 displaying unambiguous immunoreactivity along the plasma membrane and partly in the cytoplasm ( a ). The HEK cells transfected with mock displaying no intense immunoreactivity more than diffuse background fluorescence ( b ). The HEK cells transfected with TASK1 displaying no intense immunoreactivity in the absence of the primary antibody against TASK1 ( c ). Ba , Top, Voltage command pulse. Bottom, Superimposed traces of small inwardly rectifying current responses obtained at pH 6.3, 7.3, and 8.3 in the PKG-unloaded and PKGII-loaded mock-transfected HEK cells. Note no apparent pH-sensitive K + current. Bb , In the PKG-unloaded (open columns, n = 5) and PKGII-loaded (gray columns, n = 8) mock-transfected HEK cells, the mean conductances normalized to those at pH 6.3 in the current responses obtained at pH 6.3 (1.0 and 1.0, respectively), at pH 7.3 (1.2 ± 0.3 and 1.2 ± 0.2, respectively), and at pH 8.3 (1.3 ± 0.4 and 1.2 ± 0.2, respectively). C , Top, Voltage command pulses. Bottom, Sample current traces obtained at pH 6.3, 7.3, and 8.3 in TASK1-transfected HEK cells in the absence ( a ) and presence ( b ) of PKGII. Black dotted curves obtained by fitting the TASK1 currents at pH 8.3 (red traces) with GHK equation. D , Pooled data showing the mean values of normalized conductances ( a ) and the mean values of scaled conductances ( b ) at pH 6.3, 7.3, and 8.3 in the absence (open columns, n = 5) and presence (gray columns, n = 6) of PKGII. * p

    Techniques Used: Fluorescence, Transfection

    14) Product Images from "Nrf2 is controlled by two distinct ?-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity."

    Article Title: Nrf2 is controlled by two distinct ?-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity.

    Journal: Oncogene

    doi: 10.1038/onc.2012.388

    Transcription factor Nrf2 contains two separate sequences in its Neh6 domain to which β-TrCP can bind. A) COS1 cells were co-transfected with pcDNA3.1 expression plasmids encoding V5-tagged mouse Nrf2 Δ17-32 or mutants lacking SDS1, SDS2, or SDS1 and SDS2, along with pcDNA4-βTrCP1-FLAG. Empty pcDNA3.1 vector was included in the transfection mixture to normalize the amount of DNA to which cells were exposed. Following overnight transfection, the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS before whole cell lysates were prepared. An aliquot (10%) of the lysate was withdrawn as the input sample, and the remainder was used for the pull-down assay that employed an antibody against FLAG as described in Materials and Methods. B) COS1 cells were co-transfected for 24 h with an expression vector for mouse Nrf2 Δ17-32 -V5, or its mutants lacking SDSGIS 338 , SDSEME 370 and DSAPGS 378 , either individually or as double deletion mutants, along with an expression plasmid for FLAG-tagged β-TrCP1. As in panel A, β-TrCP1 was pulled-down after the cells had been subjected to 16 h serum-depletion using antibodies against FLAG, and the Nrf2 mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against the V5 epitope. C) COS1 cells were co-transfected with expression vectors for a YFP-Neh6 fusion protein, or YFP-Neh6 protein lacking SDSGIS 338 , SDSEME 370 or DSAPGS 378 , or combinations thereof, along with an expression plasmid for FLAG-tagged β-TrCP1. The Neh6 domain mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against GFP.
    Figure Legend Snippet: Transcription factor Nrf2 contains two separate sequences in its Neh6 domain to which β-TrCP can bind. A) COS1 cells were co-transfected with pcDNA3.1 expression plasmids encoding V5-tagged mouse Nrf2 Δ17-32 or mutants lacking SDS1, SDS2, or SDS1 and SDS2, along with pcDNA4-βTrCP1-FLAG. Empty pcDNA3.1 vector was included in the transfection mixture to normalize the amount of DNA to which cells were exposed. Following overnight transfection, the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS before whole cell lysates were prepared. An aliquot (10%) of the lysate was withdrawn as the input sample, and the remainder was used for the pull-down assay that employed an antibody against FLAG as described in Materials and Methods. B) COS1 cells were co-transfected for 24 h with an expression vector for mouse Nrf2 Δ17-32 -V5, or its mutants lacking SDSGIS 338 , SDSEME 370 and DSAPGS 378 , either individually or as double deletion mutants, along with an expression plasmid for FLAG-tagged β-TrCP1. As in panel A, β-TrCP1 was pulled-down after the cells had been subjected to 16 h serum-depletion using antibodies against FLAG, and the Nrf2 mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against the V5 epitope. C) COS1 cells were co-transfected with expression vectors for a YFP-Neh6 fusion protein, or YFP-Neh6 protein lacking SDSGIS 338 , SDSEME 370 or DSAPGS 378 , or combinations thereof, along with an expression plasmid for FLAG-tagged β-TrCP1. The Neh6 domain mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against GFP.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Pull Down Assay, Serum Depletion, Immunoprecipitation

    15) Product Images from "Srsf10 and the minor spliceosome control tissue-specific and dynamic SR protein expression"

    Article Title: Srsf10 and the minor spliceosome control tissue-specific and dynamic SR protein expression

    Journal: eLife

    doi: 10.7554/eLife.56075

    Srsf10 autoregulates its own splicing. ( A, B ) Confirmation of the Srsf10 knockdown. In ( A ) SRSF10 protein levels were investigated by Western Blot using an SRSF10-specific antibody. Note that both SRSF10 variants (fl and −2) are strongly reduced. The remaining signal for SRSF10-fl could indicate some unspecific detection of a different (SR)-protein. HNRNP L served as a loading control. In ( B ) confirmation of the Srsf10 knockdown on mRNA level is shown. The expression of the different Srsf10 isoforms is shown relative to Gapdh and control siRNA (n ≥ 6, mean ± SD). Note the reduced dn-E3/up-E4 ratio after knockdown of Srsf10. ( C ) Overexpression of SRSF10 variants as in Figure 1C . Top: Overexpression of the SRSF10 variants was investigated with an antibody detecting the GFP epitope. SRSF10-s was not detectably expressed. VINCULIN served as a loading control. Bottom: Representative image of splicing analysis of co-transfected minigenes. ( D ) Dose-dependent Srsf10 autoregulation. HeLa cells were cotransfected with the minigene, decreasing amounts of SRSF10-fl or −2 (0.1, 0.05, or 0.025 µg plasmid) and adjusted amounts of GFP. Expression was confirmed using an antibody against GFP (top). Note the higher expression levels of SRSF10-2. VINCULIN served as a loading control. Bottom: Splicing analysis of cotransfected minigenes (n = 2, mean ± SD). Note that barely detectable amounts of SRSF10 already change splicing with a stronger effect of SRSF10-fl, especially prominent given the lower expression level of this variant.
    Figure Legend Snippet: Srsf10 autoregulates its own splicing. ( A, B ) Confirmation of the Srsf10 knockdown. In ( A ) SRSF10 protein levels were investigated by Western Blot using an SRSF10-specific antibody. Note that both SRSF10 variants (fl and −2) are strongly reduced. The remaining signal for SRSF10-fl could indicate some unspecific detection of a different (SR)-protein. HNRNP L served as a loading control. In ( B ) confirmation of the Srsf10 knockdown on mRNA level is shown. The expression of the different Srsf10 isoforms is shown relative to Gapdh and control siRNA (n ≥ 6, mean ± SD). Note the reduced dn-E3/up-E4 ratio after knockdown of Srsf10. ( C ) Overexpression of SRSF10 variants as in Figure 1C . Top: Overexpression of the SRSF10 variants was investigated with an antibody detecting the GFP epitope. SRSF10-s was not detectably expressed. VINCULIN served as a loading control. Bottom: Representative image of splicing analysis of co-transfected minigenes. ( D ) Dose-dependent Srsf10 autoregulation. HeLa cells were cotransfected with the minigene, decreasing amounts of SRSF10-fl or −2 (0.1, 0.05, or 0.025 µg plasmid) and adjusted amounts of GFP. Expression was confirmed using an antibody against GFP (top). Note the higher expression levels of SRSF10-2. VINCULIN served as a loading control. Bottom: Splicing analysis of cotransfected minigenes (n = 2, mean ± SD). Note that barely detectable amounts of SRSF10 already change splicing with a stronger effect of SRSF10-fl, especially prominent given the lower expression level of this variant.

    Techniques Used: Western Blot, Expressing, Over Expression, Transfection, Plasmid Preparation, Variant Assay

    16) Product Images from "Cyclin B3 promotes anaphase I onset in oocyte meiosis"

    Article Title: Cyclin B3 promotes anaphase I onset in oocyte meiosis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201808091

    Interspecies cross-complementation of Ccnb3 −/− oocytes. (A) Ccnb3 −/− oocytes were injected with the indicated mRNA, induced to enter meiosis I, and scored for PB extrusion. n: number of oocytes from three independent experiments; number of oocytes analyzed and percentage of PB extrusion: 46 Ccnb3 −/− sham injected oocytes (0% PBs), 48 Ccnb3 −/− oocytes injected with mRNA coding for X. laevis cyclin B3 (85.41%), 20 Ccnb3 −/− oocytes with D. rerio cyclin B3 mRNA (60%), and 53 Ccnb3 −/− oocytes with D. melanogaster cyclin B3 mRNA (92.45%). (B) Selected time frames of collapsed z-sections (12 sections, 3-µm steps) from a representative spinning disk confocal movie of Ccnb3 −/− sham-injected oocytes, and Ccnb3 −/− oocytes injected with X. laevis cyclin B3 mRNA. Before live imaging, oocytes were incubated with SiR-DNA. Top panel shows the DIC channel and bottom panel shows siR-DNA staining in far-red. Time points after GVBD are indicated as hours:minutes. Scale bar: 20 µm. White asterisks: PBs. n: number of oocytes from three independent experiments.
    Figure Legend Snippet: Interspecies cross-complementation of Ccnb3 −/− oocytes. (A) Ccnb3 −/− oocytes were injected with the indicated mRNA, induced to enter meiosis I, and scored for PB extrusion. n: number of oocytes from three independent experiments; number of oocytes analyzed and percentage of PB extrusion: 46 Ccnb3 −/− sham injected oocytes (0% PBs), 48 Ccnb3 −/− oocytes injected with mRNA coding for X. laevis cyclin B3 (85.41%), 20 Ccnb3 −/− oocytes with D. rerio cyclin B3 mRNA (60%), and 53 Ccnb3 −/− oocytes with D. melanogaster cyclin B3 mRNA (92.45%). (B) Selected time frames of collapsed z-sections (12 sections, 3-µm steps) from a representative spinning disk confocal movie of Ccnb3 −/− sham-injected oocytes, and Ccnb3 −/− oocytes injected with X. laevis cyclin B3 mRNA. Before live imaging, oocytes were incubated with SiR-DNA. Top panel shows the DIC channel and bottom panel shows siR-DNA staining in far-red. Time points after GVBD are indicated as hours:minutes. Scale bar: 20 µm. White asterisks: PBs. n: number of oocytes from three independent experiments.

    Techniques Used: Injection, Imaging, Incubation, Staining

    Cell cycle arrest in Ccnb3 −/− oocytes is associated with incomplete APC/C activation. (A) Schematic of separase activity sensor. See text for details. (B) Failure to activate separase in the absence of cyclin B3. Separase activity sensor mRNA was injected into GV oocytes, which were released into meiosis I and visualized by spinning-disk confocal microscopy. Selected time frames of collapsed z-sections (11 sections, 3-µm steps) from a representative video are shown. Time points after GVBD are indicated as hours:minutes. Scale bar, 20 µm. White asterisks indicate PBs. n: number of oocytes from three independent experiments. (C) Western blot analysis of cyclin B1 and securin during oocyte maturation, at the time points, indicated as hours:minutes after GVBD. β-Actin served as the loading control. The number of oocytes used and the presence or absence of a PB are indicated. Two mice of each genotype were used per experiment. The data shown are representative of results from two independent experiments. (D) Total cyclin B–CDK1 activity during oocyte maturation, at the time points indicated as hours:minutes after GVBD. Histone H1 was used as a substrate. Five oocytes were used per kinase reaction; presence or absence of a PB is indicated. The graph shows quantification of phosphate incorporation from three independent experiments, and error bars indicate SD (means ± SD: lane 1: 100 [used for normalization]; lane 2: 31.27 ± 37.28; lane 3: 109.34 ± 17.09; lane 4: 104.05 ± 19.33; lane 5: 98.67 ± 50.56; lane 6: 65.40 ± 22.12). (E) CDK inhibition rescues meiosis I division. Oocytes were incubated with siR-DNA to visualize chromosomes. In metaphase I, 6 h 20 min after GVBD, oocytes were treated with 0.2 mM roscovitine (final concentration), where indicated, and the video was started. Selected time frames of collapsed z-sections (11 sections, 3-µm steps) of DIC far-red channel from a representative movie of Ccnb3 −/− oocytes with or without roscovitine treatment are shown. The asterisk indicates chromosome segregation in anaphase I. Time points after GVBD are indicated as hours:minutes. Scale bar: 20 µm. n: number of oocytes from three independent experiments. (F) Degradation of exogenous APC/C substrates. Securin-YFP (top) or cyclin B1–GFP (bottom) mRNA was injected into GV oocytes. Stills from representative videos are shown. Time points after GVBD are indicated as hours:minutes. Scale bar, 20 µm. n: number of oocytes from two independent experiment); white asterisk indicates PB extrusion (PBE). Fluorescence intensities (mean ± SD) were quantified from the indicated number of oocytes imaged (securin-YFP: 4 Ccnb3 −/− , 6 Ccnb3 +/− ; cyclin B1–GFP injections: 6 Ccnb3 −/− , 8 Ccnb3 +/− ).
    Figure Legend Snippet: Cell cycle arrest in Ccnb3 −/− oocytes is associated with incomplete APC/C activation. (A) Schematic of separase activity sensor. See text for details. (B) Failure to activate separase in the absence of cyclin B3. Separase activity sensor mRNA was injected into GV oocytes, which were released into meiosis I and visualized by spinning-disk confocal microscopy. Selected time frames of collapsed z-sections (11 sections, 3-µm steps) from a representative video are shown. Time points after GVBD are indicated as hours:minutes. Scale bar, 20 µm. White asterisks indicate PBs. n: number of oocytes from three independent experiments. (C) Western blot analysis of cyclin B1 and securin during oocyte maturation, at the time points, indicated as hours:minutes after GVBD. β-Actin served as the loading control. The number of oocytes used and the presence or absence of a PB are indicated. Two mice of each genotype were used per experiment. The data shown are representative of results from two independent experiments. (D) Total cyclin B–CDK1 activity during oocyte maturation, at the time points indicated as hours:minutes after GVBD. Histone H1 was used as a substrate. Five oocytes were used per kinase reaction; presence or absence of a PB is indicated. The graph shows quantification of phosphate incorporation from three independent experiments, and error bars indicate SD (means ± SD: lane 1: 100 [used for normalization]; lane 2: 31.27 ± 37.28; lane 3: 109.34 ± 17.09; lane 4: 104.05 ± 19.33; lane 5: 98.67 ± 50.56; lane 6: 65.40 ± 22.12). (E) CDK inhibition rescues meiosis I division. Oocytes were incubated with siR-DNA to visualize chromosomes. In metaphase I, 6 h 20 min after GVBD, oocytes were treated with 0.2 mM roscovitine (final concentration), where indicated, and the video was started. Selected time frames of collapsed z-sections (11 sections, 3-µm steps) of DIC far-red channel from a representative movie of Ccnb3 −/− oocytes with or without roscovitine treatment are shown. The asterisk indicates chromosome segregation in anaphase I. Time points after GVBD are indicated as hours:minutes. Scale bar: 20 µm. n: number of oocytes from three independent experiments. (F) Degradation of exogenous APC/C substrates. Securin-YFP (top) or cyclin B1–GFP (bottom) mRNA was injected into GV oocytes. Stills from representative videos are shown. Time points after GVBD are indicated as hours:minutes. Scale bar, 20 µm. n: number of oocytes from two independent experiment); white asterisk indicates PB extrusion (PBE). Fluorescence intensities (mean ± SD) were quantified from the indicated number of oocytes imaged (securin-YFP: 4 Ccnb3 −/− , 6 Ccnb3 +/− ; cyclin B1–GFP injections: 6 Ccnb3 −/− , 8 Ccnb3 +/− ).

    Techniques Used: Activation Assay, Activity Assay, Injection, Confocal Microscopy, Western Blot, Mouse Assay, Inhibition, Incubation, Concentration Assay, Fluorescence

    Only cyclin B3 that can support in vitro kinase activity can rescue Ccnb3 −/− oocytes. (A) Affinity purification of cyclin B3–CDK1 complexes. MBPHis cyclin B3 or MBPHis cyclin B3 MRL mutant were expressed in insect cells alone or coexpressed with either untagged or HA-tagged CDK1. Left: The eluates from purification on amylose resin were separated on SDS-PAGE and stained with Coomassie. Right: Representative autoradiograph (top) and quantification (bottom) from histone H1 kinase assays. In the graph, values in each experiment ( n = 3) were normalized to the signal from the MBPHis cyclin B3 MRL sample (lane 4 in the autoradiograph); lines indicate means (lane 1: 1.733; lane 2: 4.351; lane 3: 3.878; lane 4: 0 [used for normalization]; lane 5: 0.121; lane 6: −0.202). (B) Rescue of Ccnb3 −/− oocytes by expression of exogenous cyclin B3. Ccnb3 −/− oocytes were sham injected or injected with the indicated cyclin mRNA, then released into meiosis I. Frames of representative videos are shown. Times after GVBD are indicated as hours:minutes, and percentages of oocytes of the shown phenotypes are indicated. Scale bar, 20 µm. White asterisks: PBs. n: number of oocytes from three independent experiments. (C) Total cyclin B–CDK1 activity during oocyte maturation, in control (lane 1–3), and Ccnb3 −/− (lanes 4–9, labeled in red) oocytes expressing wild-type cyclin B3 (lanes 6 and 7) or ΔDbox cyclin B3 (lanes 8 and 9), at the time points indicated as hours:minutes after GVBD. Oocytes extruding PBs are indicated. Histone H1 was used as a substrate. A representative example from two independent experiments is shown above; quantification of both experiments is shown below ( 32 P-H1 signal normalized to the signal in lane 2; points are values from each experiment; lines indicate means: lane 1: 6.61; lane 2: 100 [used for normalization]; lane 3: 14.38; lane 4: 191.58; lane 5: 138.53; lane 6: 109.88; lane 7: 9.59; lane 8: 37.36; lane 9: 1.5). (D) Representative chromosome spreads 16 h after GVBD. Chromosomes were stained with Hoechst (blue) and kinetochores with CREST (green). Insets show typical chromosome figures observed (chromosomes are shown in grayscale). Scale bar, 5 µm. n: number of oocytes from three independent experiments. Schematics of metaphase I bivalents or metaphase II univalent chromosomes are shown to aid interpretation.
    Figure Legend Snippet: Only cyclin B3 that can support in vitro kinase activity can rescue Ccnb3 −/− oocytes. (A) Affinity purification of cyclin B3–CDK1 complexes. MBPHis cyclin B3 or MBPHis cyclin B3 MRL mutant were expressed in insect cells alone or coexpressed with either untagged or HA-tagged CDK1. Left: The eluates from purification on amylose resin were separated on SDS-PAGE and stained with Coomassie. Right: Representative autoradiograph (top) and quantification (bottom) from histone H1 kinase assays. In the graph, values in each experiment ( n = 3) were normalized to the signal from the MBPHis cyclin B3 MRL sample (lane 4 in the autoradiograph); lines indicate means (lane 1: 1.733; lane 2: 4.351; lane 3: 3.878; lane 4: 0 [used for normalization]; lane 5: 0.121; lane 6: −0.202). (B) Rescue of Ccnb3 −/− oocytes by expression of exogenous cyclin B3. Ccnb3 −/− oocytes were sham injected or injected with the indicated cyclin mRNA, then released into meiosis I. Frames of representative videos are shown. Times after GVBD are indicated as hours:minutes, and percentages of oocytes of the shown phenotypes are indicated. Scale bar, 20 µm. White asterisks: PBs. n: number of oocytes from three independent experiments. (C) Total cyclin B–CDK1 activity during oocyte maturation, in control (lane 1–3), and Ccnb3 −/− (lanes 4–9, labeled in red) oocytes expressing wild-type cyclin B3 (lanes 6 and 7) or ΔDbox cyclin B3 (lanes 8 and 9), at the time points indicated as hours:minutes after GVBD. Oocytes extruding PBs are indicated. Histone H1 was used as a substrate. A representative example from two independent experiments is shown above; quantification of both experiments is shown below ( 32 P-H1 signal normalized to the signal in lane 2; points are values from each experiment; lines indicate means: lane 1: 6.61; lane 2: 100 [used for normalization]; lane 3: 14.38; lane 4: 191.58; lane 5: 138.53; lane 6: 109.88; lane 7: 9.59; lane 8: 37.36; lane 9: 1.5). (D) Representative chromosome spreads 16 h after GVBD. Chromosomes were stained with Hoechst (blue) and kinetochores with CREST (green). Insets show typical chromosome figures observed (chromosomes are shown in grayscale). Scale bar, 5 µm. n: number of oocytes from three independent experiments. Schematics of metaphase I bivalents or metaphase II univalent chromosomes are shown to aid interpretation.

    Techniques Used: In Vitro, Activity Assay, Affinity Purification, Mutagenesis, Purification, SDS Page, Staining, Autoradiography, Expressing, Injection, Labeling

    17) Product Images from "Cloning and functional expression of a novel degenerin-like Na+ channel gene in mammals"

    Article Title: Cloning and functional expression of a novel degenerin-like Na+ channel gene in mammals

    Journal: The Journal of Physiology

    doi: 10.1111/j.1469-7793.1999.0323m.x

    Tissue distribution of BLINaC mRNA in different mouse and rat tissues A , RT-PCR experiments performed with RNA isolated from different mouse tissues (indicated on top of each lane). PCR products were hybridized with an α- 32 P-labelled mouse BLINaC cDNA probe (upper panel). The expected PCR product size is given on the left. Amplification of GAPDH was used for control (lower panel, ethidium bromide staining). B , analysis of BLINaC expression on different rat tissues (indicated on top) by RT-PCR. Specificity was confirmed by hybridization of the expected 365 bp PCR product with an α- 32 P-labelled rat BLINaC cDNA probe (upper panel). A control amplification of β-actin was performed in parallel (lower panel, ethidium bromide staining). C , detection of BLINaC transcript on mouse brain, liver and small intestine poly A + RNA (5 μg per lane) by Northern blot analysis. The probe used corresponds to an α- 32 P-labelled mouse BLINaC cDNA fragment. The filter was reprobed with a GAPDH probe as an RNA loading control (lower panel). A transcript of 2.1 kb was strongly detected in liver while a smaller transcript of 1.6 kb was found in small intestine. D , BLINaC mRNA expression in mouse liver and in freshly prepared hepatocytes was assessed by RT-PCR experiments. Specific amplification in liver and pure hepatocytes of a 737 bp product (upper panel, Southern blot) and β-actin control amplification (lower panel, ethidium bromide staining) are shown. The control corresponds to a PCR without cDNA.
    Figure Legend Snippet: Tissue distribution of BLINaC mRNA in different mouse and rat tissues A , RT-PCR experiments performed with RNA isolated from different mouse tissues (indicated on top of each lane). PCR products were hybridized with an α- 32 P-labelled mouse BLINaC cDNA probe (upper panel). The expected PCR product size is given on the left. Amplification of GAPDH was used for control (lower panel, ethidium bromide staining). B , analysis of BLINaC expression on different rat tissues (indicated on top) by RT-PCR. Specificity was confirmed by hybridization of the expected 365 bp PCR product with an α- 32 P-labelled rat BLINaC cDNA probe (upper panel). A control amplification of β-actin was performed in parallel (lower panel, ethidium bromide staining). C , detection of BLINaC transcript on mouse brain, liver and small intestine poly A + RNA (5 μg per lane) by Northern blot analysis. The probe used corresponds to an α- 32 P-labelled mouse BLINaC cDNA fragment. The filter was reprobed with a GAPDH probe as an RNA loading control (lower panel). A transcript of 2.1 kb was strongly detected in liver while a smaller transcript of 1.6 kb was found in small intestine. D , BLINaC mRNA expression in mouse liver and in freshly prepared hepatocytes was assessed by RT-PCR experiments. Specific amplification in liver and pure hepatocytes of a 737 bp product (upper panel, Southern blot) and β-actin control amplification (lower panel, ethidium bromide staining) are shown. The control corresponds to a PCR without cDNA.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Amplification, Staining, Expressing, Hybridization, Northern Blot, Southern Blot

    18) Product Images from "Ca2+-dependent Binding and Activation of Dormant Ezrin by Dimeric S100P"

    Article Title: Ca2+-dependent Binding and Activation of Dormant Ezrin by Dimeric S100P

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E02-09-0553

    Direct interaction between ezrin and S100P derivatives analyzed by affinity chromatography approaches. In each experiment, His-tagged protein (WT S100P, F15A S100P, or ezrin) was bound to the Ni-matrix, and the potential ligand (ezrin, WT S100P, F15A S100P, S100A1, or S100A11) was added as purified untagged protein in the fluid phase. Binding reactions were carried out in the presence of Ca 2 + and columns were then washed with a Ca 2 + -containing buffer. Subsequently the columns were treated with an EGTA-containing buffer to elute Ca 2 + -dependently bound proteins, and finally they were stripped with imidazole-containing buffer. Equivalent amounts of all fractions were subjected to SDS-PAGE, and proteins were visualized by Coomassie staining. To unambiguously identify the recombinantly expressed ezrin, samples of an independent experiment shown in the bottom part of panel A were analyzed by immunoblotting with antiezrin antibodies. Lanes 1 always show the flow-through, lanes 2–4 washing steps in the presence of Ca 2 + , lanes 5–7 EGTA elution steps, and lanes 8 and 9 fractions of the imidazole stripping, respectively. M, molecular weight markers. (A) Immobilized His-WT S100P with thrombin-cleaved ezrin in the fluid phase; (B) immobilized His-F15A S100P with thrombin-cleaved ezrin in the fluid phase; (C) immobilized His-ezrin with thrombin-cleaved WT S100P in the fluid phase; (D) immobilized His-ezrin with thrombin-cleaved F15A S100P in the fluid phase; (E) immobilized His-ezrin with untagged S100A1 in the fluid phase; (F) immobilized His-ezrin with untagged S100A11 in the fluid phase.
    Figure Legend Snippet: Direct interaction between ezrin and S100P derivatives analyzed by affinity chromatography approaches. In each experiment, His-tagged protein (WT S100P, F15A S100P, or ezrin) was bound to the Ni-matrix, and the potential ligand (ezrin, WT S100P, F15A S100P, S100A1, or S100A11) was added as purified untagged protein in the fluid phase. Binding reactions were carried out in the presence of Ca 2 + and columns were then washed with a Ca 2 + -containing buffer. Subsequently the columns were treated with an EGTA-containing buffer to elute Ca 2 + -dependently bound proteins, and finally they were stripped with imidazole-containing buffer. Equivalent amounts of all fractions were subjected to SDS-PAGE, and proteins were visualized by Coomassie staining. To unambiguously identify the recombinantly expressed ezrin, samples of an independent experiment shown in the bottom part of panel A were analyzed by immunoblotting with antiezrin antibodies. Lanes 1 always show the flow-through, lanes 2–4 washing steps in the presence of Ca 2 + , lanes 5–7 EGTA elution steps, and lanes 8 and 9 fractions of the imidazole stripping, respectively. M, molecular weight markers. (A) Immobilized His-WT S100P with thrombin-cleaved ezrin in the fluid phase; (B) immobilized His-F15A S100P with thrombin-cleaved ezrin in the fluid phase; (C) immobilized His-ezrin with thrombin-cleaved WT S100P in the fluid phase; (D) immobilized His-ezrin with thrombin-cleaved F15A S100P in the fluid phase; (E) immobilized His-ezrin with untagged S100A1 in the fluid phase; (F) immobilized His-ezrin with untagged S100A11 in the fluid phase.

    Techniques Used: Affinity Chromatography, Purification, Binding Assay, SDS Page, Staining, Flow Cytometry, Stripping Membranes, Molecular Weight

    Localization of ezrin and GFP-tagged WT S100P in unstimulated A431 cells. A431 cells were transiently transfected with a plasmid encoding GFP-WT S100P and were allowed to express the GFP-fusion protein for 3 d. Subsequently, the cells were fixed with PFA, permeabilized, and stained with antibodies against human ezrin (left panel). A merged image of the ezrin and GFP-S100P signals is shown in the inset. Note that both proteins show a diffuse distribution with no obvious colocalization. A substantial fraction of the GFP-S100P is present in the nucleus, most likely a consequence of the overexpression and a resulting lack of cytosolic binding partners.
    Figure Legend Snippet: Localization of ezrin and GFP-tagged WT S100P in unstimulated A431 cells. A431 cells were transiently transfected with a plasmid encoding GFP-WT S100P and were allowed to express the GFP-fusion protein for 3 d. Subsequently, the cells were fixed with PFA, permeabilized, and stained with antibodies against human ezrin (left panel). A merged image of the ezrin and GFP-S100P signals is shown in the inset. Note that both proteins show a diffuse distribution with no obvious colocalization. A substantial fraction of the GFP-S100P is present in the nucleus, most likely a consequence of the overexpression and a resulting lack of cytosolic binding partners.

    Techniques Used: Transfection, Plasmid Preparation, Staining, Over Expression, Binding Assay

    F-actin cosedimentation assays. (A) WT S100P (lanes 1–6) or F15A S100P (lanes 7–9) were mixed with ezrin and F-actin either in the presence of Ca 2 + (lanes 1–3 for WT S100P, and lanes 7–9 for F15A S100P) or in the presence of EGTA (lanes 4–6). After incubation and high-speed centrifugation the resulting supernatants were collected (lanes 1, 4, and 7). The remaining pellets were washed once with incubation buffer (lanes 2, 5, and 8 represent the supernatants of these washes), and the final pellets (lanes 3, 6, and 9) were dissolved by boiling in SDS-sample buffer. Equivalent aliquots of all fractions were subjected to SDS-PAGE. M, molecular weight markers. (B) Quantification of the cosedimentation data. Coomassie-stained gels containing the different fractions of F-actin cosedimentation experiments (one example is shown in A) were analyzed on a Lumi-Imager (Boehringer Mannheim) for quantification of the stained protein bands. For each protein, the signal obtained was normalized to the total amount of the respective protein added to reaction, which was set arbitrarily to 100%. Error bars indicate SD of the eight independent experiments analyzed. SN is the supernatant after the first centrifugation, i.e., the protein not bound to or incorporated into F-actin filaments, wash is the supernatant after washing of the F-actin pellet in incubation buffer, and P represents the protein fraction remaining in the final pellet, i.e., the proteins incorporated into (actin) or bound to (ezrin, S100P) F-actin filaments. Note the F-actin-ezrin cosedimentation in the presence of WT but not F15A S100P, which is only observed in the presence of Ca 2 + . The amount of ezrin copelleted in the absence of Ca 2 + (EGTA, lane 6) is not increased with increasing actin concentrations (in contrast to reactions performed in the presence of Ca 2 + , unpublished results) and thus most likely represents a nonspecific background. Cosedimentation experiments performed only with ezrin and F-actin reveal no appreciable ezrin pelleting, indicating that the ezrin preparation used is in the dormant conformation and not activated by mild proteolysis (B).
    Figure Legend Snippet: F-actin cosedimentation assays. (A) WT S100P (lanes 1–6) or F15A S100P (lanes 7–9) were mixed with ezrin and F-actin either in the presence of Ca 2 + (lanes 1–3 for WT S100P, and lanes 7–9 for F15A S100P) or in the presence of EGTA (lanes 4–6). After incubation and high-speed centrifugation the resulting supernatants were collected (lanes 1, 4, and 7). The remaining pellets were washed once with incubation buffer (lanes 2, 5, and 8 represent the supernatants of these washes), and the final pellets (lanes 3, 6, and 9) were dissolved by boiling in SDS-sample buffer. Equivalent aliquots of all fractions were subjected to SDS-PAGE. M, molecular weight markers. (B) Quantification of the cosedimentation data. Coomassie-stained gels containing the different fractions of F-actin cosedimentation experiments (one example is shown in A) were analyzed on a Lumi-Imager (Boehringer Mannheim) for quantification of the stained protein bands. For each protein, the signal obtained was normalized to the total amount of the respective protein added to reaction, which was set arbitrarily to 100%. Error bars indicate SD of the eight independent experiments analyzed. SN is the supernatant after the first centrifugation, i.e., the protein not bound to or incorporated into F-actin filaments, wash is the supernatant after washing of the F-actin pellet in incubation buffer, and P represents the protein fraction remaining in the final pellet, i.e., the proteins incorporated into (actin) or bound to (ezrin, S100P) F-actin filaments. Note the F-actin-ezrin cosedimentation in the presence of WT but not F15A S100P, which is only observed in the presence of Ca 2 + . The amount of ezrin copelleted in the absence of Ca 2 + (EGTA, lane 6) is not increased with increasing actin concentrations (in contrast to reactions performed in the presence of Ca 2 + , unpublished results) and thus most likely represents a nonspecific background. Cosedimentation experiments performed only with ezrin and F-actin reveal no appreciable ezrin pelleting, indicating that the ezrin preparation used is in the dormant conformation and not activated by mild proteolysis (B).

    Techniques Used: Incubation, Centrifugation, SDS Page, Molecular Weight, Staining

    19) Product Images from "Long non-coding RNA OIP5-AS1 suppresses multiple myeloma progression by sponging miR-27a-3p to activate TSC1 expression"

    Article Title: Long non-coding RNA OIP5-AS1 suppresses multiple myeloma progression by sponging miR-27a-3p to activate TSC1 expression

    Journal: Cancer Cell International

    doi: 10.1186/s12935-020-01234-7

    MiR-27a-3p could target TSC1 in MM. a The binding sites between miR-27a-3p and TSC1 3′UTR, as well as the mutant were exhibited. b Dual-luciferase reporter assay was carried out to measure the luciferase activity in TSC1-wt group and TSC1-mut group of NCI-H929 and MM1.S cells. c The levels of miR-27a-3p and TSC1 were examined after Ago2 or IgG RIP by qRT-PCR assay. d The expression of TSC1 protein in NCI-H929 and MM1.S cells transfected with NC, miR-27a-3p, anti-NC or anti-miR-27a-3p was tested by western blot assay. e The expression of TSC1 protein in NCI-H929 and MM1.S cells transfected with Lnc-NC, LncRNA OIP5-AS1, LncRNA OIP5-AS1 + NC or LncRNA OIP5-AS1 + miR-27a-3p was measured by western blot assay. f , g The mRNA and protein levels of TSC1 in bone marrows of 38 MM patients and 25 healthy donors were determined by qRT-PCR and western blot assays, respectively. h, i The correlation between the expression of TSC1 mRNA and OIP5-AS1, as well as between the expression of miR-27a-3p and TSC1 mRNA was determined via Pearson correlation analysis. * P
    Figure Legend Snippet: MiR-27a-3p could target TSC1 in MM. a The binding sites between miR-27a-3p and TSC1 3′UTR, as well as the mutant were exhibited. b Dual-luciferase reporter assay was carried out to measure the luciferase activity in TSC1-wt group and TSC1-mut group of NCI-H929 and MM1.S cells. c The levels of miR-27a-3p and TSC1 were examined after Ago2 or IgG RIP by qRT-PCR assay. d The expression of TSC1 protein in NCI-H929 and MM1.S cells transfected with NC, miR-27a-3p, anti-NC or anti-miR-27a-3p was tested by western blot assay. e The expression of TSC1 protein in NCI-H929 and MM1.S cells transfected with Lnc-NC, LncRNA OIP5-AS1, LncRNA OIP5-AS1 + NC or LncRNA OIP5-AS1 + miR-27a-3p was measured by western blot assay. f , g The mRNA and protein levels of TSC1 in bone marrows of 38 MM patients and 25 healthy donors were determined by qRT-PCR and western blot assays, respectively. h, i The correlation between the expression of TSC1 mRNA and OIP5-AS1, as well as between the expression of miR-27a-3p and TSC1 mRNA was determined via Pearson correlation analysis. * P

    Techniques Used: Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Expressing, Transfection, Western Blot

    LncRNA OIP5-AS1 inhibited tumor growth in vivo. MM xenograft model was built by injecting NCI-H929 or MM1.S cells stably expressing LncRNA OIP5-AS1 or Lnc-NC into nude mice. a , b Tumor volume and weight were recorded. c , d Levels of OIP5-AS1 and miR-27a-3p were examined by qRT-PCR assay. e Protein levels of PCNA, Cleaved caspase 3/total caspase 3 and TSC1 were measured using western blot analysis. * P
    Figure Legend Snippet: LncRNA OIP5-AS1 inhibited tumor growth in vivo. MM xenograft model was built by injecting NCI-H929 or MM1.S cells stably expressing LncRNA OIP5-AS1 or Lnc-NC into nude mice. a , b Tumor volume and weight were recorded. c , d Levels of OIP5-AS1 and miR-27a-3p were examined by qRT-PCR assay. e Protein levels of PCNA, Cleaved caspase 3/total caspase 3 and TSC1 were measured using western blot analysis. * P

    Techniques Used: In Vivo, Stable Transfection, Expressing, Mouse Assay, Quantitative RT-PCR, Western Blot

    LncRNA OIP5-AS1 was low expressed in bone marrows of MM patients. a , b The relative expression of lncRNA OIP5-AS1 in bone marrows of 38 MM patients and 25 healthy donors was tested by qRT-PCR assay. c Kaplan–Meier analysis for the prognosis of 38 MM patients was done. * P
    Figure Legend Snippet: LncRNA OIP5-AS1 was low expressed in bone marrows of MM patients. a , b The relative expression of lncRNA OIP5-AS1 in bone marrows of 38 MM patients and 25 healthy donors was tested by qRT-PCR assay. c Kaplan–Meier analysis for the prognosis of 38 MM patients was done. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Overexpression of OIP5-AS1 inhibited proliferation and metastasis, but promoted apoptosis of MM cells. MM NCI-H929 and MM1.S cells were transfected with LncRNA OIP5-AS1 or Lnc-NC. a The relative expression of lncRNA OIP5-AS1 in transfected cells was analyzed by qRT-PCR assay. b Cell viability of transfected cells was analyzed by CCK-8 assay. c The colony formation ability of transfected cells was monitored via colony formation assay. d , e The apoptosis rate and cell cycle distribution were tested through flow cytometry assay. f , g The migration and invasion abilities of transfected cells were examined by transwell assay. h , i The protein levels of Cleaved caspase 3/total caspase 3, Cleaved caspase 1, γ-H2AX, Cyclin D1, p21, Ki-67, MMP9, MMP7 and MMP10 were evaluated by western blot analysis. * P
    Figure Legend Snippet: Overexpression of OIP5-AS1 inhibited proliferation and metastasis, but promoted apoptosis of MM cells. MM NCI-H929 and MM1.S cells were transfected with LncRNA OIP5-AS1 or Lnc-NC. a The relative expression of lncRNA OIP5-AS1 in transfected cells was analyzed by qRT-PCR assay. b Cell viability of transfected cells was analyzed by CCK-8 assay. c The colony formation ability of transfected cells was monitored via colony formation assay. d , e The apoptosis rate and cell cycle distribution were tested through flow cytometry assay. f , g The migration and invasion abilities of transfected cells were examined by transwell assay. h , i The protein levels of Cleaved caspase 3/total caspase 3, Cleaved caspase 1, γ-H2AX, Cyclin D1, p21, Ki-67, MMP9, MMP7 and MMP10 were evaluated by western blot analysis. * P

    Techniques Used: Over Expression, Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Flow Cytometry, Migration, Transwell Assay, Western Blot

    Upregulation of miR-27a-3p counteracted the effects of OIP5-AS1 on the cellular behaviors of MM cells. MM NCI-H929 and MM1.S cells were transfected with Lnc-NC, LncRNA OIP5-AS1, LncRNA OIP5-AS1 + NC or LncRNA OIP5-AS1 + miR-27a-3p. a The relative expression of miR-27a-3p in transfected cells was detected by qRT-PCR assay. b Cell viability of transfected cells was analyzed by CCK-8 assay. c The colony formation ability of transfected cells was measured via colony formation assay. d , e The apoptosis rate and cell cycle distribution were detected through flow cytometry assay. f , g The migration and invasion abilities of transfected cells were evaluated by transwell assay. h , i Protein levels of Cleaved caspase 3/total caspase 3, Cleaved caspase 1, γ-H2AX, Cyclin D1, p21, Ki-67, MMP9, MMP7 and MMP10 were examined by western blot analysis. * P
    Figure Legend Snippet: Upregulation of miR-27a-3p counteracted the effects of OIP5-AS1 on the cellular behaviors of MM cells. MM NCI-H929 and MM1.S cells were transfected with Lnc-NC, LncRNA OIP5-AS1, LncRNA OIP5-AS1 + NC or LncRNA OIP5-AS1 + miR-27a-3p. a The relative expression of miR-27a-3p in transfected cells was detected by qRT-PCR assay. b Cell viability of transfected cells was analyzed by CCK-8 assay. c The colony formation ability of transfected cells was measured via colony formation assay. d , e The apoptosis rate and cell cycle distribution were detected through flow cytometry assay. f , g The migration and invasion abilities of transfected cells were evaluated by transwell assay. h , i Protein levels of Cleaved caspase 3/total caspase 3, Cleaved caspase 1, γ-H2AX, Cyclin D1, p21, Ki-67, MMP9, MMP7 and MMP10 were examined by western blot analysis. * P

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Flow Cytometry, Migration, Transwell Assay, Western Blot

    LncRNA OIP5-AS1 directly targeted miR-27a-3p. a The binding sites between OIP5-AS1 and miR-27a-3p as well as the mutant were shown. b Dual-luciferase reporter assay was carried out to measure the luciferase activity in LncRNA OIP5-AS1-wt group and LncRNA OIP5-AS1-mut group of NCI-H929 and MM1.S cells. c The levels of OIP5-AS1 and miR-27a-3p were evaluated after Ago2 or IgG RIP by qRT-PCR assay. d The expression of miR-27a-3p in NCI-H929 and MM1.S cells transfected with Lnc-NC, LncRNA OIP5-AS1, si-NC or si-LncRNA OIP5-AS1 was analyzed by qRT-PCR assay. e The relative expression of miR-27a-3p in bone marrows of 38 MM patients and 25 healthy donors was tested by qRT-PCR assay. f The correlation between the expression of OIP5-AS1 and miR-27a-3p was determined via Pearson correlation analysis. * P
    Figure Legend Snippet: LncRNA OIP5-AS1 directly targeted miR-27a-3p. a The binding sites between OIP5-AS1 and miR-27a-3p as well as the mutant were shown. b Dual-luciferase reporter assay was carried out to measure the luciferase activity in LncRNA OIP5-AS1-wt group and LncRNA OIP5-AS1-mut group of NCI-H929 and MM1.S cells. c The levels of OIP5-AS1 and miR-27a-3p were evaluated after Ago2 or IgG RIP by qRT-PCR assay. d The expression of miR-27a-3p in NCI-H929 and MM1.S cells transfected with Lnc-NC, LncRNA OIP5-AS1, si-NC or si-LncRNA OIP5-AS1 was analyzed by qRT-PCR assay. e The relative expression of miR-27a-3p in bone marrows of 38 MM patients and 25 healthy donors was tested by qRT-PCR assay. f The correlation between the expression of OIP5-AS1 and miR-27a-3p was determined via Pearson correlation analysis. * P

    Techniques Used: Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Expressing, Transfection

    20) Product Images from "The mechanism of selective kinesin inhibition by kinesin binding protein"

    Article Title: The mechanism of selective kinesin inhibition by kinesin binding protein

    Journal: bioRxiv

    doi: 10.1101/2020.07.17.208736

    Disruption of cryo-EM defined KBP-kinesin interface perturbs KBP inhibition of Kif15 and Kif1A motility in cells. (a) Schematic depiction of the inducible peroxisome motility assay, with the kinesin motor domain fused to an FRB domain and PEX fused to an FKBP domain. Addition of rapalog links FRB and FKBP and induces peroxisome movement by kinesin dimers. Expression of KBP inhibits kinesin movement, such that addition of rapalog cannot induce peroxisome transport. (b) Schematic representation of the inducible peroxisome motility assay in cells. Without rapalog or KBP, peroxisomes localize in the cell center, whereas kinesin moves towards the cell periphery. Rapalog induces peroxisome transport into the cell periphery, which is inhibited in presence of KBP. (c) Representative images of peroxisomes in COS7 cells expressing Kif15_MDC-FRB, PEX-mRFP-FKBP and HA (left panels) or HA-KBP (right panels) without and with addition of rapalog. Scale bar, 10 μm. (d, f) Quantification of the area above threshold intensity in the outer 5 μm (Kif1A_MDC) or 7.5 μm (Kif15_MDC) of the cell in cells expressing Kif15_MDC-FRB (d) or Kif1A_MDC-FRB (f), PEX-mRFP-FKBP, and HA-KBP constructs without and with rapalog treatment. Values are normalized to the condition without rapalog. Data are displayed as mean ± s.e.m. (n=28-35 cells from two independent experiments). (e, g) Quantification of the percentage of cells in which peroxisome movement is observed after rapalog treatment in cells expressing Kif15_MDC-FRB (e) or Kif1A_MDC-FRB (g), PEX-mRFP-FKBP, and HA-KBP constructs. Data are displayed as mean ± s.e.m. (n=28-35 cells from two independent experiments).
    Figure Legend Snippet: Disruption of cryo-EM defined KBP-kinesin interface perturbs KBP inhibition of Kif15 and Kif1A motility in cells. (a) Schematic depiction of the inducible peroxisome motility assay, with the kinesin motor domain fused to an FRB domain and PEX fused to an FKBP domain. Addition of rapalog links FRB and FKBP and induces peroxisome movement by kinesin dimers. Expression of KBP inhibits kinesin movement, such that addition of rapalog cannot induce peroxisome transport. (b) Schematic representation of the inducible peroxisome motility assay in cells. Without rapalog or KBP, peroxisomes localize in the cell center, whereas kinesin moves towards the cell periphery. Rapalog induces peroxisome transport into the cell periphery, which is inhibited in presence of KBP. (c) Representative images of peroxisomes in COS7 cells expressing Kif15_MDC-FRB, PEX-mRFP-FKBP and HA (left panels) or HA-KBP (right panels) without and with addition of rapalog. Scale bar, 10 μm. (d, f) Quantification of the area above threshold intensity in the outer 5 μm (Kif1A_MDC) or 7.5 μm (Kif15_MDC) of the cell in cells expressing Kif15_MDC-FRB (d) or Kif1A_MDC-FRB (f), PEX-mRFP-FKBP, and HA-KBP constructs without and with rapalog treatment. Values are normalized to the condition without rapalog. Data are displayed as mean ± s.e.m. (n=28-35 cells from two independent experiments). (e, g) Quantification of the percentage of cells in which peroxisome movement is observed after rapalog treatment in cells expressing Kif15_MDC-FRB (e) or Kif1A_MDC-FRB (g), PEX-mRFP-FKBP, and HA-KBP constructs. Data are displayed as mean ± s.e.m. (n=28-35 cells from two independent experiments).

    Techniques Used: Inhibition, Motility Assay, Expressing, Construct

    21) Product Images from "Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques"

    Article Title: Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques

    Journal: Scientific Reports

    doi: 10.1038/srep35861

    Validation of CRISPR/Cas9-mediated knock-in of EGFP at β-actin locus. ( A ) Schema represents EGFP donor, wild-type allele, and knock-in allele. Arrows indicate primers for nested genomic PCR. 5′ and 3′ targeting regions are amplified by nested PCR using primers b1-b4 and c1-c4, respectively. Blue bars indicate the boundaries between β-actin genome with identical sequence to homology arms and β-actin genome or EGFP coding sequence. ( B ) PCR amplification of 5′ targeting region using nested primers b2 and b4. ( C ) PCR amplification of 3′ targeting region using nested primers c2 and c4. ( D–G ) Representative sequence chromatograms of regions D–G in ( A ). ( H ) Validation of indel in EGFP-positive neurons. Semi-nested PCR was performed with indicated primers. Representative sequence chromatogram is shown. ( I ) Single-cell RT-PCR analysis of EGFP-β-actin mRNA in EGFP-positive neurons. The DNA fragments containing the junction between EGFP and β-actin were amplified with indicated nested primers. Representative sequence chromatogram is shown. ( J ) Single-cell RT-PCR analysis of β-actin mRNA in EGFP-positive neurons. The DNA fragments containing the sgRNA target site were amplified with indicated primers using the same samples in ( I ) as templates. Representative sequence chromatogram is shown. ( K ) Expression of full length EGFP-β-actin in mice transfected with donor, Cas9/β-actin-sgRNA, and TagRFP. The 1.9-kb DNA fragment containing the entire coding sequence of EGFP-β-actin was amplified with primers k1 and k2. For control, the β-actin fragment was amplified with primers j2 and i4. M, DNA size marker; C in ( B,C,I,J ), control EGFP-negative/TagRFP-positive cells; C in ( K ), control LacZ-sgRNA transfected brain region; KI, β-actin-sgRNA#2 transfected brain region.
    Figure Legend Snippet: Validation of CRISPR/Cas9-mediated knock-in of EGFP at β-actin locus. ( A ) Schema represents EGFP donor, wild-type allele, and knock-in allele. Arrows indicate primers for nested genomic PCR. 5′ and 3′ targeting regions are amplified by nested PCR using primers b1-b4 and c1-c4, respectively. Blue bars indicate the boundaries between β-actin genome with identical sequence to homology arms and β-actin genome or EGFP coding sequence. ( B ) PCR amplification of 5′ targeting region using nested primers b2 and b4. ( C ) PCR amplification of 3′ targeting region using nested primers c2 and c4. ( D–G ) Representative sequence chromatograms of regions D–G in ( A ). ( H ) Validation of indel in EGFP-positive neurons. Semi-nested PCR was performed with indicated primers. Representative sequence chromatogram is shown. ( I ) Single-cell RT-PCR analysis of EGFP-β-actin mRNA in EGFP-positive neurons. The DNA fragments containing the junction between EGFP and β-actin were amplified with indicated nested primers. Representative sequence chromatogram is shown. ( J ) Single-cell RT-PCR analysis of β-actin mRNA in EGFP-positive neurons. The DNA fragments containing the sgRNA target site were amplified with indicated primers using the same samples in ( I ) as templates. Representative sequence chromatogram is shown. ( K ) Expression of full length EGFP-β-actin in mice transfected with donor, Cas9/β-actin-sgRNA, and TagRFP. The 1.9-kb DNA fragment containing the entire coding sequence of EGFP-β-actin was amplified with primers k1 and k2. For control, the β-actin fragment was amplified with primers j2 and i4. M, DNA size marker; C in ( B,C,I,J ), control EGFP-negative/TagRFP-positive cells; C in ( K ), control LacZ-sgRNA transfected brain region; KI, β-actin-sgRNA#2 transfected brain region.

    Techniques Used: CRISPR, Knock-In, Polymerase Chain Reaction, Amplification, Nested PCR, Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Mouse Assay, Transfection, Marker

    CRISPR/Cas9-mediated indel mutations in EGFP-negative/TagRFP-positive neurons. ( A ) The PCR amplified DNA fragments containing the β-actin-sgRNA target site from each single neuron were analyzed by DNA sequencing. Example of sequence chromatogram is shown. In this PCR product, moderate multiple peaks appeared from middle of the sequence. ( B ) PCR product in ( A ) was cloned into plasmid and sequenced. PCR product in ( A ) carried two different indel alleles. ( C ) Indel mutations in EGFP-negative/TagRFP positive neurons. Intact wild type sequence is shown on the top. Arrowhead and arrow indicate the first ATG codon of β-actin gene and the direction of sgRNA target sequence, respectively. Slashes represent predicted cleavage site. Cell#1 carried indel mutations in both alleles (see A,B ). Cell#2-4 carried indel mutations in a single allele. ( D ) Frequency of indel mutations detected in the eleven EGFP-negative/TagRFP-positive neurons.
    Figure Legend Snippet: CRISPR/Cas9-mediated indel mutations in EGFP-negative/TagRFP-positive neurons. ( A ) The PCR amplified DNA fragments containing the β-actin-sgRNA target site from each single neuron were analyzed by DNA sequencing. Example of sequence chromatogram is shown. In this PCR product, moderate multiple peaks appeared from middle of the sequence. ( B ) PCR product in ( A ) was cloned into plasmid and sequenced. PCR product in ( A ) carried two different indel alleles. ( C ) Indel mutations in EGFP-negative/TagRFP positive neurons. Intact wild type sequence is shown on the top. Arrowhead and arrow indicate the first ATG codon of β-actin gene and the direction of sgRNA target sequence, respectively. Slashes represent predicted cleavage site. Cell#1 carried indel mutations in both alleles (see A,B ). Cell#2-4 carried indel mutations in a single allele. ( D ) Frequency of indel mutations detected in the eleven EGFP-negative/TagRFP-positive neurons.

    Techniques Used: CRISPR, Polymerase Chain Reaction, Amplification, DNA Sequencing, Sequencing, Clone Assay, Plasmid Preparation

    22) Product Images from "Biodistribution and blood clearance of plasmid DNA administered in arginine peptide complexes"

    Article Title: Biodistribution and blood clearance of plasmid DNA administered in arginine peptide complexes

    Journal: Genetic Vaccines and Therapy

    doi: 10.1186/1479-0556-9-13

    mRNA expression levels of the target gene in various organs . mRNA levels were evaluated using real time RT-PCR. Plasmid DNA (100 μg) complexed with arginine peptide at an N/P ratio of 3:1 was intraperitoneally administered to mice. Mice were sacrificed at the indicated time points, and total RNA was extracted from the organs. After preparation of cDNA, PCR amplification of luciferase and GAPDH genes was performed using the specific primers described in the Materials and Methods section. Results are expressed as means ± S.D. for at least 3 different experiments.
    Figure Legend Snippet: mRNA expression levels of the target gene in various organs . mRNA levels were evaluated using real time RT-PCR. Plasmid DNA (100 μg) complexed with arginine peptide at an N/P ratio of 3:1 was intraperitoneally administered to mice. Mice were sacrificed at the indicated time points, and total RNA was extracted from the organs. After preparation of cDNA, PCR amplification of luciferase and GAPDH genes was performed using the specific primers described in the Materials and Methods section. Results are expressed as means ± S.D. for at least 3 different experiments.

    Techniques Used: Expressing, Quantitative RT-PCR, Plasmid Preparation, Mouse Assay, Polymerase Chain Reaction, Amplification, Luciferase

    Effects of DNA dose on plasmid DNA expression after delivery in arginine/DNA complexes . Various amounts of plasmid DNA complexed with arginine peptide at an N/P ratio of 3:1 were intraperitoneally administered to mice, and mRNA levels were evaluated using real time RT-PCR. Total RNA was extracted from the organs. After preparation of cDNA, PCR amplification of luciferase and GAPDH genes was performed using the specific primers described in the Materials and Methods section. Results are expressed as means ± S.D. for at least 3 different experiments.
    Figure Legend Snippet: Effects of DNA dose on plasmid DNA expression after delivery in arginine/DNA complexes . Various amounts of plasmid DNA complexed with arginine peptide at an N/P ratio of 3:1 were intraperitoneally administered to mice, and mRNA levels were evaluated using real time RT-PCR. Total RNA was extracted from the organs. After preparation of cDNA, PCR amplification of luciferase and GAPDH genes was performed using the specific primers described in the Materials and Methods section. Results are expressed as means ± S.D. for at least 3 different experiments.

    Techniques Used: Plasmid Preparation, Expressing, Mouse Assay, Quantitative RT-PCR, Polymerase Chain Reaction, Amplification, Luciferase

    23) Product Images from "Disruption of the Sjögren-Larsson Syndrome Gene Aldh3a2 in Mice Increases Keratinocyte Growth and Retards Skin Barrier Recovery *"

    Article Title: Disruption of the Sjögren-Larsson Syndrome Gene Aldh3a2 in Mice Increases Keratinocyte Growth and Retards Skin Barrier Recovery *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.714030

    Expression profiles of ALDH3 family members in human tissues. cDNAs from human liver, kidney, and brain tissue (human MTC (multiple tissue cDNA) panels, Takara Bio) ( A ) or prepared from primary keratinocytes (undifferentiated or differentiated for 7 days; CELLnTEC) ( B ) were subjected to SYBR Green-based real-time quantitative PCR using specific primers for ALDH3A1 , ALDH3A2 , ALDH3B1 , and GAPDH . Values represent the means ± S.D. from three independent reactions. A statistically significant difference is indicated (**, p
    Figure Legend Snippet: Expression profiles of ALDH3 family members in human tissues. cDNAs from human liver, kidney, and brain tissue (human MTC (multiple tissue cDNA) panels, Takara Bio) ( A ) or prepared from primary keratinocytes (undifferentiated or differentiated for 7 days; CELLnTEC) ( B ) were subjected to SYBR Green-based real-time quantitative PCR using specific primers for ALDH3A1 , ALDH3A2 , ALDH3B1 , and GAPDH . Values represent the means ± S.D. from three independent reactions. A statistically significant difference is indicated (**, p

    Techniques Used: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction

    Enhanced proliferation and oxidative stress response in Aldh3a2 −/− keratinocytes. A , primary keratinocytes were prepared from wild-type and Aldh3a2 KO mice and grown. Cell numbers were counted by microscopic observation. For each dish, cell numbers in three randomly chosen viewing fields were counted and summed. Values represent the means ± S.D. from four independent experiments. B , keratinocytes prepared from wild-type and Aldh3a2 KO mice were subjected to a [ 3 H]thymidine uptake assay. Values represent the means ± S.D. from three independent experiments. C and D , total RNAs prepared from wild-type and Aldh3a2 KO keratinocytes were subjected to SYBR Green-based real-time quantitative RT-PCR using specific primers for Ki67 ( C ), Hmox1 , Sod1 , Gclc , Gclm , Gsta1 , and Gapdh ( D ). Values are the amount of each mRNA relative to that of Gapdh and represent the means ± S.D. for three independent experiments. Statistically significant differences are indicated (**, p
    Figure Legend Snippet: Enhanced proliferation and oxidative stress response in Aldh3a2 −/− keratinocytes. A , primary keratinocytes were prepared from wild-type and Aldh3a2 KO mice and grown. Cell numbers were counted by microscopic observation. For each dish, cell numbers in three randomly chosen viewing fields were counted and summed. Values represent the means ± S.D. from four independent experiments. B , keratinocytes prepared from wild-type and Aldh3a2 KO mice were subjected to a [ 3 H]thymidine uptake assay. Values represent the means ± S.D. from three independent experiments. C and D , total RNAs prepared from wild-type and Aldh3a2 KO keratinocytes were subjected to SYBR Green-based real-time quantitative RT-PCR using specific primers for Ki67 ( C ), Hmox1 , Sod1 , Gclc , Gclm , Gsta1 , and Gapdh ( D ). Values are the amount of each mRNA relative to that of Gapdh and represent the means ± S.D. for three independent experiments. Statistically significant differences are indicated (**, p

    Techniques Used: Mouse Assay, SYBR Green Assay, Quantitative RT-PCR

    Expression profiles of Aldh3 family members in various tissues. Total RNAs prepared from liver, kidney, retina, cornea, brain, small intestine, lung, testis, spleen, dermis, and epidermis tissue ( A and C ) and from keratinocytes kept undifferentiated or differentiated for 4 days ( B and D ) obtained from wild-type ( A–D ) and Aldh3a2 KO mice ( C and D ) were subjected to SYBR Green-based real-time quantitative RT-PCR using specific primers for Aldh3a1 , Aldh3a2 , Aldh3b1 , Aldh3b2 , Aldh3b3 , and Gapdh . Values are the amount of each mRNA relative to that of Gapdh , and represent the means ± S.D. for three independent experiments. A statistically significant difference is indicated (**, p
    Figure Legend Snippet: Expression profiles of Aldh3 family members in various tissues. Total RNAs prepared from liver, kidney, retina, cornea, brain, small intestine, lung, testis, spleen, dermis, and epidermis tissue ( A and C ) and from keratinocytes kept undifferentiated or differentiated for 4 days ( B and D ) obtained from wild-type ( A–D ) and Aldh3a2 KO mice ( C and D ) were subjected to SYBR Green-based real-time quantitative RT-PCR using specific primers for Aldh3a1 , Aldh3a2 , Aldh3b1 , Aldh3b2 , Aldh3b3 , and Gapdh . Values are the amount of each mRNA relative to that of Gapdh , and represent the means ± S.D. for three independent experiments. A statistically significant difference is indicated (**, p

    Techniques Used: Expressing, Mouse Assay, SYBR Green Assay, Quantitative RT-PCR

    24) Product Images from "Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey"

    Article Title: Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey

    Journal: British Journal of Pharmacology

    doi: 10.1038/sj.bjp.0704671

    Tissue distribution of the mouse and monkey UT receptor: (a) Tissue distribution of mouse UT receptor cDNA transcripts by RT – PCR revealed expression within cardiac and vascular (thoracic but not abdominal aorta) tissue in addition to bladder and pancreas. Trace levels of expression are also observed in skeletal muscle, oesophagus, lung and adipose tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (Lower panel) by Southern analysis using full-length UT receptor cDNA probe. (b) Tissue distributions of monkey UT receptor cDNA transcripts by RT – PCR revealed expression within heart (ventricle > atrium) and arterial blood vessels (aorta not vena cava), pancreas. Detectable levels of expression were also observed in the skeletal muscle, lung, thyroid and adrenal glands, kidney, upper portions of the gastrointestinal tract (oesophagus, stomach and small intestine but not colonic tissue) and spinal cord (but not in the cortical or cerebellar samples isolated). No detectable transcripts were derived from hepatic, bladder, adipose tissue or splenic tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (lower panel) by Southern analysis using full-length UT receptor cDNA probe.
    Figure Legend Snippet: Tissue distribution of the mouse and monkey UT receptor: (a) Tissue distribution of mouse UT receptor cDNA transcripts by RT – PCR revealed expression within cardiac and vascular (thoracic but not abdominal aorta) tissue in addition to bladder and pancreas. Trace levels of expression are also observed in skeletal muscle, oesophagus, lung and adipose tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (Lower panel) by Southern analysis using full-length UT receptor cDNA probe. (b) Tissue distributions of monkey UT receptor cDNA transcripts by RT – PCR revealed expression within heart (ventricle > atrium) and arterial blood vessels (aorta not vena cava), pancreas. Detectable levels of expression were also observed in the skeletal muscle, lung, thyroid and adrenal glands, kidney, upper portions of the gastrointestinal tract (oesophagus, stomach and small intestine but not colonic tissue) and spinal cord (but not in the cortical or cerebellar samples isolated). No detectable transcripts were derived from hepatic, bladder, adipose tissue or splenic tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (lower panel) by Southern analysis using full-length UT receptor cDNA probe.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Isolation, Derivative Assay

    Tissue distribution of the mouse and monkey U-II. (a): Tissue distribution of mouse preproU-II cDNA transcripts by RT – PCR revealed expression within heart, thoracic aorta, testes, brain, skeletal muscle, liver, kidney and spleen (upper panel). Negligible expression of preproU-II was observed in the mouse gastrointestinal tract (stomach, oesophagus, small intestine and colon), bladder, pancreas, adrenal, lung and adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel). (b) Tissue distribution of monkey preproU-II cDNA transcripts by RT – PCR revealed expression within heart (ventricle and atrium), thoracic aorta, CNS (spinal cord, cerebellum and cortex), skeletal muscle, kidney, liver and spleen (upper panel). No detectable transcripts were derived from vena cava, endocrine tissues including thyroid, pancreas and adrenal glands, lung, gastrointestinal tissue (oesophagus, stomach, small intestine, colon), bladder or adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel).
    Figure Legend Snippet: Tissue distribution of the mouse and monkey U-II. (a): Tissue distribution of mouse preproU-II cDNA transcripts by RT – PCR revealed expression within heart, thoracic aorta, testes, brain, skeletal muscle, liver, kidney and spleen (upper panel). Negligible expression of preproU-II was observed in the mouse gastrointestinal tract (stomach, oesophagus, small intestine and colon), bladder, pancreas, adrenal, lung and adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel). (b) Tissue distribution of monkey preproU-II cDNA transcripts by RT – PCR revealed expression within heart (ventricle and atrium), thoracic aorta, CNS (spinal cord, cerebellum and cortex), skeletal muscle, kidney, liver and spleen (upper panel). No detectable transcripts were derived from vena cava, endocrine tissues including thyroid, pancreas and adrenal glands, lung, gastrointestinal tissue (oesophagus, stomach, small intestine, colon), bladder or adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Derivative Assay

    25) Product Images from "Enhanced Clathrin-Dependent Endocytosis in the Absence of Calnexin"

    Article Title: Enhanced Clathrin-Dependent Endocytosis in the Absence of Calnexin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021678

    Calnexin interacts with SGIP1. ( A ) A schematic representation of full length SGIP1. The N-terminal domain of SGIP1 (amino acid residues 1–239) represents a membrane phospholipid-binding domain (MP, dark green); middle domain (amino acid residues 239–653) contains a proline-rich region (Pro-Rich, light green); the C-terminal region contains an adaptor-complex-subunit (Adap-Comp-Sub, red). ( B ) N1E-115 cells were transfected with GFP-SGIP1 or GFP expression vectors and lysed cells were subjected to immunoprecipitation with anti-GFP. Calnexin was identified by Western blot (WB) analysis of imunoprecipitates probed with anti-calnexin antibodies ( lanes 3 – 6 ). Lane 1 , Western blot analysis of N1E-115 cells expressing GFP-SGIP1; lane 2 , Western blot analysis of N1E-115 cells expressing GFP; lane 3 , identification of calnexin in N1E-115 cells expressing GFP-SGIP1; lane 4 , identification of calnexin in N1E-115 cells expressing GFP; lane 5 , lysate from N1E-115 cells expressing GFP-SGIP1 were immunoprecipitated with anti-GFP antibodies followed by Western blot analysis with anti-calnexin antibodies; lane 6 , lysate from N1E-115 cells expressing GFP were immunoprecipitated with anti-GFP antibodies followed by Western blot analysis with anti-calnexin antibodies. ( C ) Immunoprecipitation of SGIP1-calnexin complexes from mouse cerebellum. Homogenate from wild-type mouse cerebellum was incubated with anti-SGIP1 antibodies or anti-His-tag antibodies followed by Western blot analysis with either anti-SGIP1 antibodies ( lanes 1 – 3 ) or anti-calnexin antibodies ( lanes 4 – 6 ). Lanes 1 and 4 , cerebellum homogenate; lanes 2 and 5 , immunoprecipitated with anti-SGIP1 antibodies; lanes 3 and 6 , immunoprecipitation with anti-His-tag antibodies. CNX, calnexin. ( D ) Immunolocalization of calnexin and GFP-SGIP1 in N1E-115 cells. N1E-115 cells were transfected with pEGFP-SGIP1 expression vector followed by confocal analysis of intracellular localization of GFP-SGIP1 and calnexin as described under “Experimental Procedures”. Scale bar = 17 µm. ( E ) mRNA were isolated from liver, brain, spinal cord and cerebellum of wild-type ( wt ) and calnexin-deficient ( cnx − / − ) mice followed by RT-PCR with DNA primers specific for SGIP1 ( SGIP1 ) or tubulin ( tubulin ) as described under “Experimental Procedures”. ( F ) Western blot analysis of protein extracts isolated from wild-type ( wt ) and calnexin-deficient ( cnx − / − ) granule cells was carried out with anti-SGIP1-antibodies as described under “Experimental Procedures”. ( G ) Yeast cells were transformed with pGBKT7 vector containing cDNA encoding calnexin C-tail ( CNX C-tail ) and pGADT7 vector or pGADT7 vector containing cDNA encoding SGIP1 or SGIP1 domains as depicted in the Figure. Yeast serial dilution-culture (10 −1 ∼10 −4 ) on SD/-Leu/-Trp plate shows both pGBKT7 and pGADT7 vectors were transformed into the AH109 strain. Yeast culture on SD/-Leu/-Trp/-His/-Ade shows yeast-two-hybrid interaction and activation of the reporter gene. Filter lift assays indicates interactions between the calnexin C-tail and SGIP1 and specifically the SGIP1 C-terminal Adap-Comp-Sub domain.
    Figure Legend Snippet: Calnexin interacts with SGIP1. ( A ) A schematic representation of full length SGIP1. The N-terminal domain of SGIP1 (amino acid residues 1–239) represents a membrane phospholipid-binding domain (MP, dark green); middle domain (amino acid residues 239–653) contains a proline-rich region (Pro-Rich, light green); the C-terminal region contains an adaptor-complex-subunit (Adap-Comp-Sub, red). ( B ) N1E-115 cells were transfected with GFP-SGIP1 or GFP expression vectors and lysed cells were subjected to immunoprecipitation with anti-GFP. Calnexin was identified by Western blot (WB) analysis of imunoprecipitates probed with anti-calnexin antibodies ( lanes 3 – 6 ). Lane 1 , Western blot analysis of N1E-115 cells expressing GFP-SGIP1; lane 2 , Western blot analysis of N1E-115 cells expressing GFP; lane 3 , identification of calnexin in N1E-115 cells expressing GFP-SGIP1; lane 4 , identification of calnexin in N1E-115 cells expressing GFP; lane 5 , lysate from N1E-115 cells expressing GFP-SGIP1 were immunoprecipitated with anti-GFP antibodies followed by Western blot analysis with anti-calnexin antibodies; lane 6 , lysate from N1E-115 cells expressing GFP were immunoprecipitated with anti-GFP antibodies followed by Western blot analysis with anti-calnexin antibodies. ( C ) Immunoprecipitation of SGIP1-calnexin complexes from mouse cerebellum. Homogenate from wild-type mouse cerebellum was incubated with anti-SGIP1 antibodies or anti-His-tag antibodies followed by Western blot analysis with either anti-SGIP1 antibodies ( lanes 1 – 3 ) or anti-calnexin antibodies ( lanes 4 – 6 ). Lanes 1 and 4 , cerebellum homogenate; lanes 2 and 5 , immunoprecipitated with anti-SGIP1 antibodies; lanes 3 and 6 , immunoprecipitation with anti-His-tag antibodies. CNX, calnexin. ( D ) Immunolocalization of calnexin and GFP-SGIP1 in N1E-115 cells. N1E-115 cells were transfected with pEGFP-SGIP1 expression vector followed by confocal analysis of intracellular localization of GFP-SGIP1 and calnexin as described under “Experimental Procedures”. Scale bar = 17 µm. ( E ) mRNA were isolated from liver, brain, spinal cord and cerebellum of wild-type ( wt ) and calnexin-deficient ( cnx − / − ) mice followed by RT-PCR with DNA primers specific for SGIP1 ( SGIP1 ) or tubulin ( tubulin ) as described under “Experimental Procedures”. ( F ) Western blot analysis of protein extracts isolated from wild-type ( wt ) and calnexin-deficient ( cnx − / − ) granule cells was carried out with anti-SGIP1-antibodies as described under “Experimental Procedures”. ( G ) Yeast cells were transformed with pGBKT7 vector containing cDNA encoding calnexin C-tail ( CNX C-tail ) and pGADT7 vector or pGADT7 vector containing cDNA encoding SGIP1 or SGIP1 domains as depicted in the Figure. Yeast serial dilution-culture (10 −1 ∼10 −4 ) on SD/-Leu/-Trp plate shows both pGBKT7 and pGADT7 vectors were transformed into the AH109 strain. Yeast culture on SD/-Leu/-Trp/-His/-Ade shows yeast-two-hybrid interaction and activation of the reporter gene. Filter lift assays indicates interactions between the calnexin C-tail and SGIP1 and specifically the SGIP1 C-terminal Adap-Comp-Sub domain.

    Techniques Used: Binding Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Incubation, Plasmid Preparation, Isolation, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Serial Dilution, Activation Assay

    26) Product Images from "The AMPA receptor-associated protein Shisa7 regulates hippocampal synaptic function and contextual memory"

    Article Title: The AMPA receptor-associated protein Shisa7 regulates hippocampal synaptic function and contextual memory

    Journal: eLife

    doi: 10.7554/eLife.24192

    Shisa7 gene expression. ( a ) Quantitative PCR shows that the Shisa7 gene expression is specifically enriched within the brain (note the log 2 -scale), and is virtually absent in the pancreas (pooled from three adult mice). For comparison, gene expression of Shisa9 and Shisa6 was taken along. Primers are indicated in Figure 1—source data 1 . ( b ) Representative example of Shisa7 in situ hybridization signal (picture 6, probe RP_050609_04_H05) from the Allen Brain Atlas ( Nesvizhskii et al., 2003 ; Lein et al., 2007 ; Wenger and Coon, 2013 ) showing Shisa7 expression in the entire forebrain, with high expression in the hippocampus, and no expression in the cerebellum. Specific brain regions are indicated. ( c ) Hippocampal gene expression of Shisa7 and AMPAR subunits Gria1 and Gria2 increases during postnatal development and stabilizes after ~3 weeks, as measured by quantitative PCR (n = 3 independent biological samples, pooled from two mice). ( d ) RT-PCR on cDNA generated from hippocampal RNA using primers flanking exon 4 of Shisa7 ( Figure 1—source data 1 ). Using this RT-PCR on WT mice in duplicate, we observed two bands (425, 375 nts) corresponding to the two Shisa7 transcripts, of which the exon 4-less transcript showed highest expression. Sequence analysis of these PCR products confirmed the presence of exon 4 (black letters) between exons 3 and 5 (gray letters) in the Shisa7 sequence. The amino acid sequence is indicated above the nucleotide sequence. Importantly, the Shisa7 tryptic peptides NLYNTMKPSNLDNHYNVNSPK (derived from exon 3 and 4) was identified using mass spectrometric analysis of native hippocampal Shisa7 complexes ( Table 1 ).
    Figure Legend Snippet: Shisa7 gene expression. ( a ) Quantitative PCR shows that the Shisa7 gene expression is specifically enriched within the brain (note the log 2 -scale), and is virtually absent in the pancreas (pooled from three adult mice). For comparison, gene expression of Shisa9 and Shisa6 was taken along. Primers are indicated in Figure 1—source data 1 . ( b ) Representative example of Shisa7 in situ hybridization signal (picture 6, probe RP_050609_04_H05) from the Allen Brain Atlas ( Nesvizhskii et al., 2003 ; Lein et al., 2007 ; Wenger and Coon, 2013 ) showing Shisa7 expression in the entire forebrain, with high expression in the hippocampus, and no expression in the cerebellum. Specific brain regions are indicated. ( c ) Hippocampal gene expression of Shisa7 and AMPAR subunits Gria1 and Gria2 increases during postnatal development and stabilizes after ~3 weeks, as measured by quantitative PCR (n = 3 independent biological samples, pooled from two mice). ( d ) RT-PCR on cDNA generated from hippocampal RNA using primers flanking exon 4 of Shisa7 ( Figure 1—source data 1 ). Using this RT-PCR on WT mice in duplicate, we observed two bands (425, 375 nts) corresponding to the two Shisa7 transcripts, of which the exon 4-less transcript showed highest expression. Sequence analysis of these PCR products confirmed the presence of exon 4 (black letters) between exons 3 and 5 (gray letters) in the Shisa7 sequence. The amino acid sequence is indicated above the nucleotide sequence. Importantly, the Shisa7 tryptic peptides NLYNTMKPSNLDNHYNVNSPK (derived from exon 3 and 4) was identified using mass spectrometric analysis of native hippocampal Shisa7 complexes ( Table 1 ).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Generated, Sequencing, Polymerase Chain Reaction, Derivative Assay

    27) Product Images from "Recessive mutations in muscle-specific isoforms of FXR1 cause congenital multi-minicore myopathy"

    Article Title: Recessive mutations in muscle-specific isoforms of FXR1 cause congenital multi-minicore myopathy

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08548-9

    Expression and subcellular localization of P82,84 isoforms in exon-15 mice. a Relative Fxr1 mRNA quantification in gastrocnemius of 2.5-months old mice by qRT-PCR using two different TaqMan probes: Fxr1-523_m1 (exons 2-3; Mm00484523_m1) and Fxr1-304_m1 (exons 14-15; Mm01286304_m1). Values are normalized to Tbp mRNA levels and represented as fold change of the mean value of wt mice. n = 6 (wt), 3(delACAG), 3(dupA). Data are mean ± s.d. followed by one-way ANOVA (***) with Tukey post-hoc test. b Representative FXR1P immunoblot of protein extracts from gastrocnemius (soluble (SF) and insoluble (IF) fractions) of delACAG and dupA mice and corresponding wt littermates. Vinculin and α-actinin were used as loading controls (2–3 months old mice, n = 4). Brackets denote isoforms e and f and non-specific bands are labeled with asterisks. c Maximum Z-project and magnification of confocal anti-FXR1P immunofluorescence images (green) in isolated EDL fibers. n = 8(wt), 3(wt/delACAG), 8(delACAG), 6(dupA). Scale bars 5 µm. FXR1P isoforms are localized in a striated pattern except in delACAG fibers where are found predominantly in granules. Heterozygous wt/delACAG fibers contain a small number of granules (arrowheads) and the fluorescent intensity of FXR1P is reduced in dupA mice. Nuclei are labeled with propidium iodide (PI, red)
    Figure Legend Snippet: Expression and subcellular localization of P82,84 isoforms in exon-15 mice. a Relative Fxr1 mRNA quantification in gastrocnemius of 2.5-months old mice by qRT-PCR using two different TaqMan probes: Fxr1-523_m1 (exons 2-3; Mm00484523_m1) and Fxr1-304_m1 (exons 14-15; Mm01286304_m1). Values are normalized to Tbp mRNA levels and represented as fold change of the mean value of wt mice. n = 6 (wt), 3(delACAG), 3(dupA). Data are mean ± s.d. followed by one-way ANOVA (***) with Tukey post-hoc test. b Representative FXR1P immunoblot of protein extracts from gastrocnemius (soluble (SF) and insoluble (IF) fractions) of delACAG and dupA mice and corresponding wt littermates. Vinculin and α-actinin were used as loading controls (2–3 months old mice, n = 4). Brackets denote isoforms e and f and non-specific bands are labeled with asterisks. c Maximum Z-project and magnification of confocal anti-FXR1P immunofluorescence images (green) in isolated EDL fibers. n = 8(wt), 3(wt/delACAG), 8(delACAG), 6(dupA). Scale bars 5 µm. FXR1P isoforms are localized in a striated pattern except in delACAG fibers where are found predominantly in granules. Heterozygous wt/delACAG fibers contain a small number of granules (arrowheads) and the fluorescent intensity of FXR1P is reduced in dupA mice. Nuclei are labeled with propidium iodide (PI, red)

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Labeling, Immunofluorescence, Isolation

    28) Product Images from "Divergent roles of endothelial NF-?B in multiple organ injury and bacterial clearance in mouse models of sepsis"

    Article Title: Divergent roles of endothelial NF-?B in multiple organ injury and bacterial clearance in mouse models of sepsis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20071393

    I-κBαmt mRNA expression in whole blood cells of EC-rtTA/I-κBαmt mice. Mice 1 and 2 (Ms1 and Ms2) were not fed with Dox, and mice 3–5 (Ms3, Ms4, and Ms5) were fed with Dox for 4 d. RT-PCR detected no I-κBαmt expression in whole blood cells and lungs of Ms1 and Ms2 (without Dox), and detected a strong I-κBαmt band in lungs but not in whole blood cells of Ms3, Ms4, and Ms5 (with Dox). GAPDH serves as internal control. Bld, whole blood cells; Lug, lungs; M, DNA marker; P, positive control.
    Figure Legend Snippet: I-κBαmt mRNA expression in whole blood cells of EC-rtTA/I-κBαmt mice. Mice 1 and 2 (Ms1 and Ms2) were not fed with Dox, and mice 3–5 (Ms3, Ms4, and Ms5) were fed with Dox for 4 d. RT-PCR detected no I-κBαmt expression in whole blood cells and lungs of Ms1 and Ms2 (without Dox), and detected a strong I-κBαmt band in lungs but not in whole blood cells of Ms3, Ms4, and Ms5 (with Dox). GAPDH serves as internal control. Bld, whole blood cells; Lug, lungs; M, DNA marker; P, positive control.

    Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Positive Control

    Generation of double TG EC-rtTA/I-κBαmt mice. (A) Schematic representation of VeCadrtTA and TreI-κBαmt transgenes. Transactivator (rtTA) expression is controlled by the endothelial-specific promoter VE–cadherin-5 (Ve-cad). Human I-κBαmt gene expression is controlled by a TRE-CMV fusion promoter, whose activation requires the binding of rtTA and Dox. (B and C) RT-PCR photograph showing Dox-induced I-κBαmt mRNA expression in EC-rtTA/I-κBαmt TG mice. Mouse TV616 was fed with Dox for 4 d, and mouse TV614, a transgene-positive littermate of TV616, was not fed with Dox. RT-PCR analysis detected I-κBαmt mRNA expression in 11 out of the 12 organs from mouse TV616 (B) but detected no I-κBαmt expression in any organ from mouse TV614 (C). GAPDH serves as internal control. Aot, aorta; Brn, brain; Ht, heart; Itn, intestine; Kid, kidney; Liv, liver; Lug, lungs; M, DNA marker; N, negative control; P, positive control; Skm, skeletal muscle; Spl, spleen; Stam, stomach; Thyd, thyroid; Ton, tongue. (D–F) Immunofluorescence staining for Dox-induced I-κBαmt protein expression in lung sections of EC-rtTA/I-κBαmt mice. (D) Dox + mice, preimmune IgG, no staining. (E) Dox − mice, anti–human I-κBα, weak staining (endogenous mouse I-κBα). (F) Dox + mice, anti–human I-κBα, stronger staining (Dox-induced I-κBαmt protein). Bars, 100 μm.
    Figure Legend Snippet: Generation of double TG EC-rtTA/I-κBαmt mice. (A) Schematic representation of VeCadrtTA and TreI-κBαmt transgenes. Transactivator (rtTA) expression is controlled by the endothelial-specific promoter VE–cadherin-5 (Ve-cad). Human I-κBαmt gene expression is controlled by a TRE-CMV fusion promoter, whose activation requires the binding of rtTA and Dox. (B and C) RT-PCR photograph showing Dox-induced I-κBαmt mRNA expression in EC-rtTA/I-κBαmt TG mice. Mouse TV616 was fed with Dox for 4 d, and mouse TV614, a transgene-positive littermate of TV616, was not fed with Dox. RT-PCR analysis detected I-κBαmt mRNA expression in 11 out of the 12 organs from mouse TV616 (B) but detected no I-κBαmt expression in any organ from mouse TV614 (C). GAPDH serves as internal control. Aot, aorta; Brn, brain; Ht, heart; Itn, intestine; Kid, kidney; Liv, liver; Lug, lungs; M, DNA marker; N, negative control; P, positive control; Skm, skeletal muscle; Spl, spleen; Stam, stomach; Thyd, thyroid; Ton, tongue. (D–F) Immunofluorescence staining for Dox-induced I-κBαmt protein expression in lung sections of EC-rtTA/I-κBαmt mice. (D) Dox + mice, preimmune IgG, no staining. (E) Dox − mice, anti–human I-κBα, weak staining (endogenous mouse I-κBα). (F) Dox + mice, anti–human I-κBα, stronger staining (Dox-induced I-κBαmt protein). Bars, 100 μm.

    Techniques Used: Mouse Assay, Expressing, Activation Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Negative Control, Positive Control, Immunofluorescence, Staining

    29) Product Images from "Involvement of an alternatively spliced mitochondrial oxodicarboxylate carrier in adipogenesis in 3T3-L1 cells"

    Article Title: Involvement of an alternatively spliced mitochondrial oxodicarboxylate carrier in adipogenesis in 3T3-L1 cells

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-16-92

    RT-PCR analysis of ODC-AS expression in 3T3-L1 cells . Expression of ODC-AS and ODC mRNA in 3T3-L1 Cells were detected with RT-PCR in mRNAs prepared at the indicated days from 3T3-L1 cell pre- and after differentiation, and visualized under UV after agarose gel electrophoresis with ethidium bromide. 1 kb DNA standards is shown on the right. The addition of differentiation reagents was indicated at the bottom. G3PDH was amplified as an internal control and shown below. L: 1 kb DNA ladder; PC: positive control for ODC from cloned cDNA plasmid; NC: negative control for G3PDH (water).
    Figure Legend Snippet: RT-PCR analysis of ODC-AS expression in 3T3-L1 cells . Expression of ODC-AS and ODC mRNA in 3T3-L1 Cells were detected with RT-PCR in mRNAs prepared at the indicated days from 3T3-L1 cell pre- and after differentiation, and visualized under UV after agarose gel electrophoresis with ethidium bromide. 1 kb DNA standards is shown on the right. The addition of differentiation reagents was indicated at the bottom. G3PDH was amplified as an internal control and shown below. L: 1 kb DNA ladder; PC: positive control for ODC from cloned cDNA plasmid; NC: negative control for G3PDH (water).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Amplification, Positive Control, Clone Assay, Plasmid Preparation, Negative Control

    ODC and ODC-AS expression in fat tissues . A. ODC-AS gene expression in fat tissues by cold exposure. ODC-AS expression in brown adipose tissue (BAT) and white adipose tissue (WAT) as quantified by real-time PCR and expressed as the fold increase of G3PDH, after 18 hour exposure of mice at 4°C and room temperature. Data shown are means ± S.D, n = 5. * indicates P
    Figure Legend Snippet: ODC and ODC-AS expression in fat tissues . A. ODC-AS gene expression in fat tissues by cold exposure. ODC-AS expression in brown adipose tissue (BAT) and white adipose tissue (WAT) as quantified by real-time PCR and expressed as the fold increase of G3PDH, after 18 hour exposure of mice at 4°C and room temperature. Data shown are means ± S.D, n = 5. * indicates P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    ODC-AS knockdown and qRT-PCR of marker genes in adipogenesis . 3T3-L1 preadipocytes were infected with lentivirus (Nsi, ODCi-3 or ODCi-5) before the induction for differentiation. Twenty four hours after induction, the cells were collected and examined for mRNA levels of PPARγ, C/EBPα, aP2 and CD36 (see Methods). Data were expressed as means ± SEM (n = 3), relative to Nsi. *compared with Nsi: P
    Figure Legend Snippet: ODC-AS knockdown and qRT-PCR of marker genes in adipogenesis . 3T3-L1 preadipocytes were infected with lentivirus (Nsi, ODCi-3 or ODCi-5) before the induction for differentiation. Twenty four hours after induction, the cells were collected and examined for mRNA levels of PPARγ, C/EBPα, aP2 and CD36 (see Methods). Data were expressed as means ± SEM (n = 3), relative to Nsi. *compared with Nsi: P

    Techniques Used: Quantitative RT-PCR, Marker, Infection

    30) Product Images from "A neonatal murine model for evaluation of enterovirus E HY12 virus infection and pathogenicity"

    Article Title: A neonatal murine model for evaluation of enterovirus E HY12 virus infection and pathogenicity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193155

    Minimal infective dose of HY12 to ICR suckling mice. 2 × 10 4 , 2 × 10 6 and 2 × 10 8 TCID 50 HY12 viruses were injected to mice subcutaneously to determine the minimal infective dose (MID). Tissue samples were collected 5 dpi and processed for RT-PCR to detect the virus genome fragment. Representative figure showing the PCR-amplified fragments with expected size from mice infected with 2 × 10 6 TCID 50 (lane 2) and 2 × 10 8 TCID 50 (lane 3), respectively. The negative and positive controls were presented in lane 4 and lane 5, respectively. M stands for the DNA ladder.
    Figure Legend Snippet: Minimal infective dose of HY12 to ICR suckling mice. 2 × 10 4 , 2 × 10 6 and 2 × 10 8 TCID 50 HY12 viruses were injected to mice subcutaneously to determine the minimal infective dose (MID). Tissue samples were collected 5 dpi and processed for RT-PCR to detect the virus genome fragment. Representative figure showing the PCR-amplified fragments with expected size from mice infected with 2 × 10 6 TCID 50 (lane 2) and 2 × 10 8 TCID 50 (lane 3), respectively. The negative and positive controls were presented in lane 4 and lane 5, respectively. M stands for the DNA ladder.

    Techniques Used: Mouse Assay, Injection, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Infection

    ICR suckling mice are susceptible to HY12 enterovirus infection. Three-day old Balb/c, Kunming, and IRC neonatal mice were subcutaneously inoculated with 2×10 6 TCID 50 HY12 viruses. Tissue samples including liver, lung, spleen, lymph node, kidney, and brain were collected from pups at 5 dpi and processed for amplification of HY12 genomic fragments using RT-PCR. Fragment with expected size was detected clearly from tissue samples in ICR (lane 2) suckling mice. No fragments were amplified from Balb/C suckling mice (lane 1) and Kunming suckling mice (lane 3) infected with HY12. Lane 4 and lane 5 were the positive and negative control, respectively. DNA ladder was presented as M and the size of expected fragment is indicated as arrow.
    Figure Legend Snippet: ICR suckling mice are susceptible to HY12 enterovirus infection. Three-day old Balb/c, Kunming, and IRC neonatal mice were subcutaneously inoculated with 2×10 6 TCID 50 HY12 viruses. Tissue samples including liver, lung, spleen, lymph node, kidney, and brain were collected from pups at 5 dpi and processed for amplification of HY12 genomic fragments using RT-PCR. Fragment with expected size was detected clearly from tissue samples in ICR (lane 2) suckling mice. No fragments were amplified from Balb/C suckling mice (lane 1) and Kunming suckling mice (lane 3) infected with HY12. Lane 4 and lane 5 were the positive and negative control, respectively. DNA ladder was presented as M and the size of expected fragment is indicated as arrow.

    Techniques Used: Mouse Assay, Infection, Amplification, Reverse Transcription Polymerase Chain Reaction, Negative Control

    31) Product Images from "Mesothelin Is Not Required for Normal Mouse Development or Reproduction"

    Article Title: Mesothelin Is Not Required for Normal Mouse Development or Reproduction

    Journal: Molecular and Cellular Biology

    doi:

    Expression of mesothelin transcript in mutant null mice. (A) Northern blot analysis of the mesothelin transcript. Total RNA was prepared from lung tissues of wild-type (+/+) and mutant (−/−) mice. RNA (20 μg from each) was separated on a 1.2% agarose formaldehyde gel and hybridized with a mouse cDNA mesothelin probe covering most of the mesothelin coding sequence (top). Bottom, methylene blue staining of total RNA transferred onto the nylon membrane shown at the top prior to hybridization. (B) RT-PCR analysis of the mesothelin transcript. RNAs from wild type and mutant mice were reverse transcribed, amplified by PCR using the primer pair T75 and T76 (see Materials and Methods), and analyzed in 2% agarose gel.
    Figure Legend Snippet: Expression of mesothelin transcript in mutant null mice. (A) Northern blot analysis of the mesothelin transcript. Total RNA was prepared from lung tissues of wild-type (+/+) and mutant (−/−) mice. RNA (20 μg from each) was separated on a 1.2% agarose formaldehyde gel and hybridized with a mouse cDNA mesothelin probe covering most of the mesothelin coding sequence (top). Bottom, methylene blue staining of total RNA transferred onto the nylon membrane shown at the top prior to hybridization. (B) RT-PCR analysis of the mesothelin transcript. RNAs from wild type and mutant mice were reverse transcribed, amplified by PCR using the primer pair T75 and T76 (see Materials and Methods), and analyzed in 2% agarose gel.

    Techniques Used: Expressing, Mutagenesis, Mouse Assay, Northern Blot, Sequencing, Staining, Hybridization, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    32) Product Images from "HIF-driven SF3B1 induces KHK-C to enforce fructolysis and heart disease"

    Article Title: HIF-driven SF3B1 induces KHK-C to enforce fructolysis and heart disease

    Journal: Nature

    doi: 10.1038/nature14508

    Downstream metabolic effects of fructose metabolism in NMCs a , log 2 fold change of metabolites in NMCs overexpressing KHK-C and cultured with glucose or glucose and fructose compared to the corresponding control transduced NMCs ( n = 4 biological replicates per group). b , Incorporation of [ 3 H]fructose into RNA (top), DNA (middle) and protein (bottom) of NMCs treated as indicated ( n = 4 biological replicates per group). c , Uptake of [ 14 C]deoxyglucose in NMCs infected as in b at depicted time points ( n = 4 biological replicates per group). d , [ 3 H]leucine incorporation in NMCs transduced with shNs or shKhk and co-transduced with either empty overexpression vector or KHK-A or KHK-C respectively. NMCs were cultured in media with increasing fructose concentrations, under physiologic glucose amounts or with increasing glucose concentrations under physiologic fructose amounts. Data are presented relative to shNs/empty-vector-transduced NMCs at 5 mM glucose/0 μM fructose ( n = 4 biological replicates). e , ADP/ATP ratios in NMCs transduced as indicated. ( n = 4 biological replicates, data show 1 of 3 representative experiments). f , Immunofluorescence images of NMCs transduced with empty vector (upper panel) or HIF1αΔODD (middle panel) lentiviruses or exposed to hypoxia (lower panel) were additionally transduced as indicated. Prior to staining for sarcomeric α-actinin, Oil Red O and DAPI, NMCs were incubated with oleic acid. g , Quantification of lipid droplets/cell in NMCs of immunofluorescent images shown in f . h , Ratio of lipid droplets/cell area of NMCs shown in g . i–l , NMCs were infected as indicated and processed for oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements under basal conditions or after injection of OA or FCCP. Depicted are rates expressed as OCR to ECAR ratios (upper panels) or individual ECAR (for measurements at baseline) or OCR values ( n = 8 biological replicates for HIF1αΔODD/shKhk-A; n = 10 biological replicates for all other groups). Error bars are s.d. ( b–e ) or s.e.m. ( g–l ). * P
    Figure Legend Snippet: Downstream metabolic effects of fructose metabolism in NMCs a , log 2 fold change of metabolites in NMCs overexpressing KHK-C and cultured with glucose or glucose and fructose compared to the corresponding control transduced NMCs ( n = 4 biological replicates per group). b , Incorporation of [ 3 H]fructose into RNA (top), DNA (middle) and protein (bottom) of NMCs treated as indicated ( n = 4 biological replicates per group). c , Uptake of [ 14 C]deoxyglucose in NMCs infected as in b at depicted time points ( n = 4 biological replicates per group). d , [ 3 H]leucine incorporation in NMCs transduced with shNs or shKhk and co-transduced with either empty overexpression vector or KHK-A or KHK-C respectively. NMCs were cultured in media with increasing fructose concentrations, under physiologic glucose amounts or with increasing glucose concentrations under physiologic fructose amounts. Data are presented relative to shNs/empty-vector-transduced NMCs at 5 mM glucose/0 μM fructose ( n = 4 biological replicates). e , ADP/ATP ratios in NMCs transduced as indicated. ( n = 4 biological replicates, data show 1 of 3 representative experiments). f , Immunofluorescence images of NMCs transduced with empty vector (upper panel) or HIF1αΔODD (middle panel) lentiviruses or exposed to hypoxia (lower panel) were additionally transduced as indicated. Prior to staining for sarcomeric α-actinin, Oil Red O and DAPI, NMCs were incubated with oleic acid. g , Quantification of lipid droplets/cell in NMCs of immunofluorescent images shown in f . h , Ratio of lipid droplets/cell area of NMCs shown in g . i–l , NMCs were infected as indicated and processed for oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements under basal conditions or after injection of OA or FCCP. Depicted are rates expressed as OCR to ECAR ratios (upper panels) or individual ECAR (for measurements at baseline) or OCR values ( n = 8 biological replicates for HIF1αΔODD/shKhk-A; n = 10 biological replicates for all other groups). Error bars are s.d. ( b–e ) or s.e.m. ( g–l ). * P

    Techniques Used: Cell Culture, Infection, Transduction, Over Expression, Plasmid Preparation, Immunofluorescence, Staining, Incubation, Injection

    33) Product Images from "Essential role for InSyn1 in dystroglycan complex integrity and cognitive behaviors in mice"

    Article Title: Essential role for InSyn1 in dystroglycan complex integrity and cognitive behaviors in mice

    Journal: eLife

    doi: 10.7554/eLife.50712

    Generation of InSyn1 KO mice. ( A ) Multiple protein sequence alignment of InSyn1 from human ( Homo sapiens ), rat ( Rattus norvegicus ), mouse ( Mus musculus ), Dog ( Canis lupus ), Wild pig ( Sus scrofa ), monkey ( Macaca fascicularis ), marmoset ( Callithrix jacchus ), chicken ( Gallus gallus ), Lamprey ( Petromyzon marinus ), platypus ( Ornithorhynchus anatinus ), xenopus ( Xenopus laevis ), and two fish species ( Takifugu rubripes ), ( Danio rerio ), The set of sequences were chosen from the Gene Tree of human InSyn1 (ENSGT00910000144204). The sequences were aligned by multiple sequence alignment algorithm Kalign2 and manually curated by JalView ( Lassmann et al., 2009 ). The consensus sequence with sequence logos is depicted below. The predicted coiled-coil region and the N terminus 60 amino acid-deleted InSyn1 mutant (InSyn1ΔN) are indicated above. Note the sequence similarities of InSyn1 between different species. ( B ) Schematic of InSyn1 gene structure (top, gray box regions representing coding sequence) and the alignment of DNA sequencing from WT (top trace) and InSyn1 (bottom trace) KO mice showing an 11bp-deletion in exon two in InSyn1 KO mice. ( C ) Representative images of PCR-based genotyping. F; a common forward primer. R1 and R5; reverse primers depicted in B. ( D and E ). HITI labeling of endogenous InSyn1 in WT and KO hippocampal neurons. ( D ) WT neurons exhibit clear puncta staining along the neurite while InSyn1 null neurons lost puncta staining. scale bars, 10 µm. ( E ) InSyn1 puncta-positive cells were quantified from three different samples. Of note, no InSyn1-positive cells were detected from null hippocampal neurons.
    Figure Legend Snippet: Generation of InSyn1 KO mice. ( A ) Multiple protein sequence alignment of InSyn1 from human ( Homo sapiens ), rat ( Rattus norvegicus ), mouse ( Mus musculus ), Dog ( Canis lupus ), Wild pig ( Sus scrofa ), monkey ( Macaca fascicularis ), marmoset ( Callithrix jacchus ), chicken ( Gallus gallus ), Lamprey ( Petromyzon marinus ), platypus ( Ornithorhynchus anatinus ), xenopus ( Xenopus laevis ), and two fish species ( Takifugu rubripes ), ( Danio rerio ), The set of sequences were chosen from the Gene Tree of human InSyn1 (ENSGT00910000144204). The sequences were aligned by multiple sequence alignment algorithm Kalign2 and manually curated by JalView ( Lassmann et al., 2009 ). The consensus sequence with sequence logos is depicted below. The predicted coiled-coil region and the N terminus 60 amino acid-deleted InSyn1 mutant (InSyn1ΔN) are indicated above. Note the sequence similarities of InSyn1 between different species. ( B ) Schematic of InSyn1 gene structure (top, gray box regions representing coding sequence) and the alignment of DNA sequencing from WT (top trace) and InSyn1 (bottom trace) KO mice showing an 11bp-deletion in exon two in InSyn1 KO mice. ( C ) Representative images of PCR-based genotyping. F; a common forward primer. R1 and R5; reverse primers depicted in B. ( D and E ). HITI labeling of endogenous InSyn1 in WT and KO hippocampal neurons. ( D ) WT neurons exhibit clear puncta staining along the neurite while InSyn1 null neurons lost puncta staining. scale bars, 10 µm. ( E ) InSyn1 puncta-positive cells were quantified from three different samples. Of note, no InSyn1-positive cells were detected from null hippocampal neurons.

    Techniques Used: Mouse Assay, Sequencing, Fluorescence In Situ Hybridization, Mutagenesis, DNA Sequencing, Polymerase Chain Reaction, Labeling, Staining

    34) Product Images from "A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells"

    Article Title: A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054500

    Roles for G proteins in Sweeteners Effects on Differentiation of 3T3-L1 cells. A. Expression profiles of Gαgust, Gα14 and Gαs during differentiation of 3T3-L1 cells. The total RNAs were prepared from 3T3-L1 cells as described in Fig. 1 and the mRNA levels of Gαgust, Gα14 and Gαs were measured by quantitative RT-PCR using mouse ribosomal protein S18 as an internal control. Results are shown as the mean ± SE (n = 3–6). B. 3T3-L1 cells were differentiated without (control) or with sucralose (20 mM), saccharin (20 mM), or endothelin-1 (20 nM) in the absence (0.1% DMSO) or the presence of YM-254890 (10 µM). The expression levels of PPARγ and C/EBPα at Day 2 (48 hours) were measured by immunoblotting. Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Gray and black bars show the control and the plus YM-254890 data, respectively. Results are shown as the mean values from two independent experiments. C. Undifferentiated 3T3-L1 cells were detached and transfected with the expression vectors containing wild-type or G226A mutant Gαs cDNAs (20 μg each) by electroporation as described in ‘ Materials and Methods ’. Transfected cells were seeded on a 6-well culture dish and cultured to confluence before induction of differentiation without (control) or with sucralose (20 mM) or saccharin (20 mM). The expression levels of PPARγ and C/EBPα were measured by immunoblotting at Day 2 (48 hours). Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Gray and black bars show the control and the Gαs-G226A data, respectively. Results are shown as the mean ± SE (n = 3). P
    Figure Legend Snippet: Roles for G proteins in Sweeteners Effects on Differentiation of 3T3-L1 cells. A. Expression profiles of Gαgust, Gα14 and Gαs during differentiation of 3T3-L1 cells. The total RNAs were prepared from 3T3-L1 cells as described in Fig. 1 and the mRNA levels of Gαgust, Gα14 and Gαs were measured by quantitative RT-PCR using mouse ribosomal protein S18 as an internal control. Results are shown as the mean ± SE (n = 3–6). B. 3T3-L1 cells were differentiated without (control) or with sucralose (20 mM), saccharin (20 mM), or endothelin-1 (20 nM) in the absence (0.1% DMSO) or the presence of YM-254890 (10 µM). The expression levels of PPARγ and C/EBPα at Day 2 (48 hours) were measured by immunoblotting. Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Gray and black bars show the control and the plus YM-254890 data, respectively. Results are shown as the mean values from two independent experiments. C. Undifferentiated 3T3-L1 cells were detached and transfected with the expression vectors containing wild-type or G226A mutant Gαs cDNAs (20 μg each) by electroporation as described in ‘ Materials and Methods ’. Transfected cells were seeded on a 6-well culture dish and cultured to confluence before induction of differentiation without (control) or with sucralose (20 mM) or saccharin (20 mM). The expression levels of PPARγ and C/EBPα were measured by immunoblotting at Day 2 (48 hours). Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Gray and black bars show the control and the Gαs-G226A data, respectively. Results are shown as the mean ± SE (n = 3). P

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Mutagenesis, Electroporation, Cell Culture

    35) Product Images from "Nrf2 is controlled by two distinct ?-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity."

    Article Title: Nrf2 is controlled by two distinct ?-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity.

    Journal: Oncogene

    doi: 10.1038/onc.2012.388

    Nrf2 is down-regulated by prevention of the inhibitory phosphorylation of GSK-3 A) COS1 cells were transfected with a pcDNA3.1 expression vector for a V5-tagged fusion protein comprising Neh6(LacZ)-V5 or Neh6(LacZ)-V5 bearing individual deletion of the SDSGIS 338 , SDSEME 370 and DSAPGS 378 from the Neh6 domain for 24 h. Twenty-four h later the cells were serum depleted by transfer to DMEM containing 0.1% (v/v) FBS for a further 16 h before the cells were treated with either 10 μM LY294002, 5 μM MK-2206 or with vehicle (0.1% (v/v) DMSO) in media containing 0.1% (w/v) FBS for 8 h. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immuno-blotted with the indicated antibodies. The antibody that recognised phospho-GSK-3β (Ser-9) was from Abcam (ab30619). Gapdh was used as an internal control. B) Keap1 −/− MEFs were seeded in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for a further 16 h. Thereafter, the cells were treated for 8 h with 1.0, 2.5, 10 or 40 μM LY294002 or 0.25, 1.0, 5.0 or 10 μM MK-2206, all of which were dissolved in DMSO to a final concentration of 0.1% (by vol), in media containing 0.1% (w/v) FBS; 0.1% (v/v) DMSO was used as vehicle control. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immunoblotted with the indicated antibodies. Gapdh was used as a sample loading control. C) Keap1 −/− MEFs were grown in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for 16 h, as described in panel B above. The fibroblasts were then treated with LY294002 or MK-2206 in medium containing 0.1% FBS, at the doses indicated, for 2 h before they were transferred to fresh medium containing 0.1% FBS for 6 h. Thereafter, the fibroblasts were harvested, total RNA extracted, and mRNA for Nqo1, Hmox1, Gclc, Gclm, Gsta1 and Gstm1 measured by TaqMan chemistry as described by Higgins et al ( 61 ). The solid horizontal bar indicates that mRNA levels in MEFs treated with kinase inhibitors in medium containing 0.1% FBS were compared with MEFs treated with DMSO vehicle control in medium containing 0.1% FBS.
    Figure Legend Snippet: Nrf2 is down-regulated by prevention of the inhibitory phosphorylation of GSK-3 A) COS1 cells were transfected with a pcDNA3.1 expression vector for a V5-tagged fusion protein comprising Neh6(LacZ)-V5 or Neh6(LacZ)-V5 bearing individual deletion of the SDSGIS 338 , SDSEME 370 and DSAPGS 378 from the Neh6 domain for 24 h. Twenty-four h later the cells were serum depleted by transfer to DMEM containing 0.1% (v/v) FBS for a further 16 h before the cells were treated with either 10 μM LY294002, 5 μM MK-2206 or with vehicle (0.1% (v/v) DMSO) in media containing 0.1% (w/v) FBS for 8 h. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immuno-blotted with the indicated antibodies. The antibody that recognised phospho-GSK-3β (Ser-9) was from Abcam (ab30619). Gapdh was used as an internal control. B) Keap1 −/− MEFs were seeded in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for a further 16 h. Thereafter, the cells were treated for 8 h with 1.0, 2.5, 10 or 40 μM LY294002 or 0.25, 1.0, 5.0 or 10 μM MK-2206, all of which were dissolved in DMSO to a final concentration of 0.1% (by vol), in media containing 0.1% (w/v) FBS; 0.1% (v/v) DMSO was used as vehicle control. Whole-cell lysates were harvested and proteins were resolved in SDS-PAGE and gels were immunoblotted with the indicated antibodies. Gapdh was used as a sample loading control. C) Keap1 −/− MEFs were grown in 60 mm petri-dishes in DMEM containing 10% FBS 24 h prior to serum depletion (0.1% FBS) for 16 h, as described in panel B above. The fibroblasts were then treated with LY294002 or MK-2206 in medium containing 0.1% FBS, at the doses indicated, for 2 h before they were transferred to fresh medium containing 0.1% FBS for 6 h. Thereafter, the fibroblasts were harvested, total RNA extracted, and mRNA for Nqo1, Hmox1, Gclc, Gclm, Gsta1 and Gstm1 measured by TaqMan chemistry as described by Higgins et al ( 61 ). The solid horizontal bar indicates that mRNA levels in MEFs treated with kinase inhibitors in medium containing 0.1% FBS were compared with MEFs treated with DMSO vehicle control in medium containing 0.1% FBS.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, SDS Page, Serum Depletion, Concentration Assay

    β-TrCP binds both phosphorylated and non-phosphorylated Nrf2-derived peptides containing the DSGIS and DSAPGS sequences. Biotinylated-peptides, designed around sequences in the SDS1 and SDS2 regions of the Neh6 domain in mouse Nrf2, were coupled with Streptavidin, which had first been immobilized on agarose beads, and used in pull-down assays to identify those that were bound by in vitro translated [ 35 S]methionine-labelled β-TrCP1. A biotinylated ETGE-containing peptide, representing residues 73-90 of mouse Nrf2, was used as a negative control. A) Ala-scanning substitutions were introduced into the (SGSG)MEFN DSDSGISLN TSPSR peptide between residues equivalent to Asp-332 and Asn-340 in the Neh6 domain (the residues changed are shown underlined). Each of the peptides was used in the pull-down assay, and autoradiography was used to identify β-TrCP1 that bound the peptides. As a control, lane 1 contained total [ 35 S]methionine-labelled in vitro translated protein. Lane 2 shows [ 35 S]methionine-labelled protein pulled-down by the ETGE peptide. Lane 3 shows [ 35 S]methionine-labelled protein pulled-down by the wild-type 22-mer peptide, and lanes 4-12 show protein pulled down by the peptides with Ala substitutions across residues 332-340. B) Ala-scanning substitutions were introduced into the (SGSG)SEME ELDS A PGSVK QNGP peptide between residues equivalent to Glu-371 and Ser-374 and Pro-376 and Lys-380 in the Neh6 domain (the residues changed are shown underlined). As above, lane 1 represents total [ 35 S]methionine-labelled in vitro translated protein, lane 2 shows protein pulled-down by the ETGE peptide, lane 3 shows protein pulled-down by the wild-type 22-mer peptide. Lanes 4-7 show [ 35 S]methionine-labelled protein pulled down by the peptides with Ala substitutions across residues 371-374, and lanes 8-12 show protein pulled down by peptide with Ala substitutions across residues 376-380. C) The biotinylated-peptide pull-down assay was used to test whether double phosphorylation of the peptides increased their binding by β-TrCP; the phosphorylated residues are indicated in bold italics in the peptide shown above the gel. Lane 1 represents total [ 35 S]methionine-labelled in vitro translated protein, and lane 2 shows protein pulled down by the ETGE-containing peptide based on the Neh2 domain. Lanes 3 and 4 show the protein pulled-down by DSGIS- and DpSGIpS-containing peptides. Lanes 5 and 6 show that the SDSEME- and pSDpSEME-containing peptides did not pull-down protein. Lanes 7 and 8 show the protein pulled-down by DSAPGS- and DpSAPGpS-containing peptides. D) The effect of individual phosphorylation of Ser-333, Ser-335, Ser-338 and Ser-342 across the DSGIS-containing peptide on binding by β-TrCP was examined by pull-down assay. Lane 1 shows total [ 35 S]methionine-labelled in vitro translated protein, and lane 2 shows protein pulled-down by the ETGE-containing peptide based on the Neh2 domain. Lanes 3 and 9 show protein bound to the non-phosphorylated DSGIS-containing peptide. Lanes 4-8 show the protein bound to the DSGIS-containing peptide in which only Ser-335 is phosphorylated, only Ser-338 is phosphorylated, both Ser-335 and Ser-338 are phosphorylated, Ser-333, Ser-335 and Ser-338 are phosphorylated, and Ser-335, Ser-338 and Ser-342 had been phosphorylated, respectively.
    Figure Legend Snippet: β-TrCP binds both phosphorylated and non-phosphorylated Nrf2-derived peptides containing the DSGIS and DSAPGS sequences. Biotinylated-peptides, designed around sequences in the SDS1 and SDS2 regions of the Neh6 domain in mouse Nrf2, were coupled with Streptavidin, which had first been immobilized on agarose beads, and used in pull-down assays to identify those that were bound by in vitro translated [ 35 S]methionine-labelled β-TrCP1. A biotinylated ETGE-containing peptide, representing residues 73-90 of mouse Nrf2, was used as a negative control. A) Ala-scanning substitutions were introduced into the (SGSG)MEFN DSDSGISLN TSPSR peptide between residues equivalent to Asp-332 and Asn-340 in the Neh6 domain (the residues changed are shown underlined). Each of the peptides was used in the pull-down assay, and autoradiography was used to identify β-TrCP1 that bound the peptides. As a control, lane 1 contained total [ 35 S]methionine-labelled in vitro translated protein. Lane 2 shows [ 35 S]methionine-labelled protein pulled-down by the ETGE peptide. Lane 3 shows [ 35 S]methionine-labelled protein pulled-down by the wild-type 22-mer peptide, and lanes 4-12 show protein pulled down by the peptides with Ala substitutions across residues 332-340. B) Ala-scanning substitutions were introduced into the (SGSG)SEME ELDS A PGSVK QNGP peptide between residues equivalent to Glu-371 and Ser-374 and Pro-376 and Lys-380 in the Neh6 domain (the residues changed are shown underlined). As above, lane 1 represents total [ 35 S]methionine-labelled in vitro translated protein, lane 2 shows protein pulled-down by the ETGE peptide, lane 3 shows protein pulled-down by the wild-type 22-mer peptide. Lanes 4-7 show [ 35 S]methionine-labelled protein pulled down by the peptides with Ala substitutions across residues 371-374, and lanes 8-12 show protein pulled down by peptide with Ala substitutions across residues 376-380. C) The biotinylated-peptide pull-down assay was used to test whether double phosphorylation of the peptides increased their binding by β-TrCP; the phosphorylated residues are indicated in bold italics in the peptide shown above the gel. Lane 1 represents total [ 35 S]methionine-labelled in vitro translated protein, and lane 2 shows protein pulled down by the ETGE-containing peptide based on the Neh2 domain. Lanes 3 and 4 show the protein pulled-down by DSGIS- and DpSGIpS-containing peptides. Lanes 5 and 6 show that the SDSEME- and pSDpSEME-containing peptides did not pull-down protein. Lanes 7 and 8 show the protein pulled-down by DSAPGS- and DpSAPGpS-containing peptides. D) The effect of individual phosphorylation of Ser-333, Ser-335, Ser-338 and Ser-342 across the DSGIS-containing peptide on binding by β-TrCP was examined by pull-down assay. Lane 1 shows total [ 35 S]methionine-labelled in vitro translated protein, and lane 2 shows protein pulled-down by the ETGE-containing peptide based on the Neh2 domain. Lanes 3 and 9 show protein bound to the non-phosphorylated DSGIS-containing peptide. Lanes 4-8 show the protein bound to the DSGIS-containing peptide in which only Ser-335 is phosphorylated, only Ser-338 is phosphorylated, both Ser-335 and Ser-338 are phosphorylated, Ser-333, Ser-335 and Ser-338 are phosphorylated, and Ser-335, Ser-338 and Ser-342 had been phosphorylated, respectively.

    Techniques Used: Derivative Assay, In Vitro, Negative Control, Pull Down Assay, Autoradiography, Binding Assay

    Repression of Nrf2 by β-TrCP occurs in both a GSK-3-dependent and a GSK-3-independent manner Nrf2 is subject to dual regulation by Keap1 and β-TrCP. The cartoon shows that Nrf2 is repressed by Keap1 though DLG and ETGE motifs in its Neh2 domain, both of which are required for ubiquitylation of the CNC-bZIP protein by Cul3-Rbx1. By contrast, Nrf2 is repressed by β-TrCP though DSGIS and DSAPGS motifs in its Neh6 domain, each of which is sufficient for ubiquitylation of the CNC-bZIP protein by Cul1-Rbx1. Phosphorylation of the DSGIS motif increases its degron activity, and this is positively regulated by GSK-3. The GSK-3 inhibitor CT99021 decreases the degron activity of the DSGIS destruction motif whereas PI3K and PKB/Akt inhibitors increase the degron activity of the DSGIS motif. By contrast, the DSAPGS destruction motif is not influenced by GSK-3 activity.
    Figure Legend Snippet: Repression of Nrf2 by β-TrCP occurs in both a GSK-3-dependent and a GSK-3-independent manner Nrf2 is subject to dual regulation by Keap1 and β-TrCP. The cartoon shows that Nrf2 is repressed by Keap1 though DLG and ETGE motifs in its Neh2 domain, both of which are required for ubiquitylation of the CNC-bZIP protein by Cul3-Rbx1. By contrast, Nrf2 is repressed by β-TrCP though DSGIS and DSAPGS motifs in its Neh6 domain, each of which is sufficient for ubiquitylation of the CNC-bZIP protein by Cul1-Rbx1. Phosphorylation of the DSGIS motif increases its degron activity, and this is positively regulated by GSK-3. The GSK-3 inhibitor CT99021 decreases the degron activity of the DSGIS destruction motif whereas PI3K and PKB/Akt inhibitors increase the degron activity of the DSGIS motif. By contrast, the DSAPGS destruction motif is not influenced by GSK-3 activity.

    Techniques Used: Activity Assay

    Repression of Nrf2 by β-TrCP is abolished by deletion of two separate regions within its Neh6 domain. A) Keap1 −/− MEFs were transfected for 24 h with pcDNA3.1 expression vectors encoding V5-tagged mouse Nrf2 ΔNeh2 or related mutants that lack SDS1, PEST or the entire Neh6 domain, along with either an empty pcDNA4-FLAG plasmid or a pcDNA4-βTrCP1-FLAG plasmid. Twenty-four h later, the cells were serum-depleted by transfer to Delbecco’s modified Eagle’s medium (DMEM) containing 0.5% fetal bovine serum (FBS) for 16 h, after which whole cell lysates were prepared and ectopic Nrf2 measured by Western blotting using mouse anti-V5 antibodies. B) Keap1 −/− MEFs were transfected with the same Nrf2 ΔNeh2 -V5, Nrf2 ΔNeh2,SDS1 -V5, Nrf2 ΔNeh2,PEST -V5 and Nrf2 ΔNeh2,Neh6 -V5 expression vectors used in panel A. Following transfection, the MEFs were serum-depleted for 16 h before they were treated with CHX for various periods of time and the relative amounts of ectopic mutant Nrf2 measured by Western blotting. Results that were significantly higher than the Nrf2 ΔNeh2 control with P values of 0.01-0.001 or
    Figure Legend Snippet: Repression of Nrf2 by β-TrCP is abolished by deletion of two separate regions within its Neh6 domain. A) Keap1 −/− MEFs were transfected for 24 h with pcDNA3.1 expression vectors encoding V5-tagged mouse Nrf2 ΔNeh2 or related mutants that lack SDS1, PEST or the entire Neh6 domain, along with either an empty pcDNA4-FLAG plasmid or a pcDNA4-βTrCP1-FLAG plasmid. Twenty-four h later, the cells were serum-depleted by transfer to Delbecco’s modified Eagle’s medium (DMEM) containing 0.5% fetal bovine serum (FBS) for 16 h, after which whole cell lysates were prepared and ectopic Nrf2 measured by Western blotting using mouse anti-V5 antibodies. B) Keap1 −/− MEFs were transfected with the same Nrf2 ΔNeh2 -V5, Nrf2 ΔNeh2,SDS1 -V5, Nrf2 ΔNeh2,PEST -V5 and Nrf2 ΔNeh2,Neh6 -V5 expression vectors used in panel A. Following transfection, the MEFs were serum-depleted for 16 h before they were treated with CHX for various periods of time and the relative amounts of ectopic mutant Nrf2 measured by Western blotting. Results that were significantly higher than the Nrf2 ΔNeh2 control with P values of 0.01-0.001 or

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Modification, Western Blot, Mutagenesis

    Transcription factor Nrf2 contains two separate sequences in its Neh6 domain to which β-TrCP can bind. A) COS1 cells were co-transfected with pcDNA3.1 expression plasmids encoding V5-tagged mouse Nrf2 Δ17-32 or mutants lacking SDS1, SDS2, or SDS1 and SDS2, along with pcDNA4-βTrCP1-FLAG. Empty pcDNA3.1 vector was included in the transfection mixture to normalize the amount of DNA to which cells were exposed. Following overnight transfection, the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS before whole cell lysates were prepared. An aliquot (10%) of the lysate was withdrawn as the input sample, and the remainder was used for the pull-down assay that employed an antibody against FLAG as described in Materials and Methods. B) COS1 cells were co-transfected for 24 h with an expression vector for mouse Nrf2 Δ17-32 -V5, or its mutants lacking SDSGIS 338 , SDSEME 370 and DSAPGS 378 , either individually or as double deletion mutants, along with an expression plasmid for FLAG-tagged β-TrCP1. As in panel A, β-TrCP1 was pulled-down after the cells had been subjected to 16 h serum-depletion using antibodies against FLAG, and the Nrf2 mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against the V5 epitope. C) COS1 cells were co-transfected with expression vectors for a YFP-Neh6 fusion protein, or YFP-Neh6 protein lacking SDSGIS 338 , SDSEME 370 or DSAPGS 378 , or combinations thereof, along with an expression plasmid for FLAG-tagged β-TrCP1. The Neh6 domain mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against GFP.
    Figure Legend Snippet: Transcription factor Nrf2 contains two separate sequences in its Neh6 domain to which β-TrCP can bind. A) COS1 cells were co-transfected with pcDNA3.1 expression plasmids encoding V5-tagged mouse Nrf2 Δ17-32 or mutants lacking SDS1, SDS2, or SDS1 and SDS2, along with pcDNA4-βTrCP1-FLAG. Empty pcDNA3.1 vector was included in the transfection mixture to normalize the amount of DNA to which cells were exposed. Following overnight transfection, the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS before whole cell lysates were prepared. An aliquot (10%) of the lysate was withdrawn as the input sample, and the remainder was used for the pull-down assay that employed an antibody against FLAG as described in Materials and Methods. B) COS1 cells were co-transfected for 24 h with an expression vector for mouse Nrf2 Δ17-32 -V5, or its mutants lacking SDSGIS 338 , SDSEME 370 and DSAPGS 378 , either individually or as double deletion mutants, along with an expression plasmid for FLAG-tagged β-TrCP1. As in panel A, β-TrCP1 was pulled-down after the cells had been subjected to 16 h serum-depletion using antibodies against FLAG, and the Nrf2 mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against the V5 epitope. C) COS1 cells were co-transfected with expression vectors for a YFP-Neh6 fusion protein, or YFP-Neh6 protein lacking SDSGIS 338 , SDSEME 370 or DSAPGS 378 , or combinations thereof, along with an expression plasmid for FLAG-tagged β-TrCP1. The Neh6 domain mutants that co-immunoprecipitated with β-TrCP1 were detected by immunoblotting with antibodies against GFP.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Pull Down Assay, Serum Depletion, Immunoprecipitation

    A peptide sequence in the SDS1 region of the Neh6 domain allows Nrf2 to interact with β-TrCP in a GSK-3-dependent manner. A) COS1 cells were co-transfected with expression vectors encoding the Gal4 DNA-binding domain fused to the Neh6 domain (i.e. Gal4(DBD)-Neh6), or expression vectors for Gal4(DBD)-Neh6 containing individual or combined deletion of the SDSGIS 338 , SDSEME 370 and DSAPGS 378 hexapeptides along with an expression vector for the Gal4 activating domain fused to the substrate-binding WD40 domain of β-TrCP1 (Gal4(AD)-WD40) and the reporter plasmids P TK UAS-Luc and pRL-TK Renilla . Forty-eight h later the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS, after which time Gal4-driven luciferase activity was measured. Significant increases in Gal4-driven reporter gene activity, relative to that produced by pM-Neh6 alone, with P values of 0.01-0.001 or
    Figure Legend Snippet: A peptide sequence in the SDS1 region of the Neh6 domain allows Nrf2 to interact with β-TrCP in a GSK-3-dependent manner. A) COS1 cells were co-transfected with expression vectors encoding the Gal4 DNA-binding domain fused to the Neh6 domain (i.e. Gal4(DBD)-Neh6), or expression vectors for Gal4(DBD)-Neh6 containing individual or combined deletion of the SDSGIS 338 , SDSEME 370 and DSAPGS 378 hexapeptides along with an expression vector for the Gal4 activating domain fused to the substrate-binding WD40 domain of β-TrCP1 (Gal4(AD)-WD40) and the reporter plasmids P TK UAS-Luc and pRL-TK Renilla . Forty-eight h later the cells were serum-depleted for 16 h by transfer to DMEM containing 0.5% FBS, after which time Gal4-driven luciferase activity was measured. Significant increases in Gal4-driven reporter gene activity, relative to that produced by pM-Neh6 alone, with P values of 0.01-0.001 or

    Techniques Used: Sequencing, Transfection, Expressing, Binding Assay, Plasmid Preparation, Luciferase, Activity Assay, Produced

    β-TrCP-mediated ubiquitylation of Nrf2 involves two separate peptide motifs in the Neh6 domain A) COS1 cells were co-transfected for 24 h with a pcDNA3.1 expression vector encoding V5-tagged mouse Nrf2 Δ17-32 , or mutants lacking SDSGIS 338 , SDSEME 370 or DSAPGS 378 , along with expression plasmids for HisUb and β-TrCP1-FLAG. As controls, the cells were transfected with empty expression vectors, pHisUb alone or pcDNA3.1-Nrf2 Δ17-32 -V5 without pcDNA4-βTrCP1-FLAG. Following transfection, the cells were serum-depleted for 16 h, after which whole cell lysates were prepared in phosphate-buffered saline. To allow loading to be assessed, a 10% portion of the lysate was retained as input. The remainder of each sample was used to purify His-tagged protein separately using Ni 2+ -agarose beads, and the total amount of ubiquitylated Nrf2 protein in each sample was determined by Western blotting with anti-V5 antibodies. The input samples were also immunoblotted with anti-V5 and anti-FLAG antibodies to confirm equal loading of Nrf2 and β-TrCP1. B) The same ubiquitylation assay was performed for Nrf2 Δ17-32 -V5, Nrf2 Δ17-32,SDSGIS -V5 and Nrf2 Δ17-32,DSAPGS -V5 as in panel A, but on this occasion the Nrf2 expression constructs were co-transfected into COS1 cells with an expression vector for either GSK-3β Δ9 or an empty vector. C) The same ubiquitylation assay was performed for Nrf2 Δ17-32 -V5, Nrf2 Δ17-32,SDSGIS -V5 and Nrf2 Δ17-32,DSAPGS -V5 as in panel A, but in this case the COS1 cells were treated with 5 μM CT99021 to inhibit GSK-3.
    Figure Legend Snippet: β-TrCP-mediated ubiquitylation of Nrf2 involves two separate peptide motifs in the Neh6 domain A) COS1 cells were co-transfected for 24 h with a pcDNA3.1 expression vector encoding V5-tagged mouse Nrf2 Δ17-32 , or mutants lacking SDSGIS 338 , SDSEME 370 or DSAPGS 378 , along with expression plasmids for HisUb and β-TrCP1-FLAG. As controls, the cells were transfected with empty expression vectors, pHisUb alone or pcDNA3.1-Nrf2 Δ17-32 -V5 without pcDNA4-βTrCP1-FLAG. Following transfection, the cells were serum-depleted for 16 h, after which whole cell lysates were prepared in phosphate-buffered saline. To allow loading to be assessed, a 10% portion of the lysate was retained as input. The remainder of each sample was used to purify His-tagged protein separately using Ni 2+ -agarose beads, and the total amount of ubiquitylated Nrf2 protein in each sample was determined by Western blotting with anti-V5 antibodies. The input samples were also immunoblotted with anti-V5 and anti-FLAG antibodies to confirm equal loading of Nrf2 and β-TrCP1. B) The same ubiquitylation assay was performed for Nrf2 Δ17-32 -V5, Nrf2 Δ17-32,SDSGIS -V5 and Nrf2 Δ17-32,DSAPGS -V5 as in panel A, but on this occasion the Nrf2 expression constructs were co-transfected into COS1 cells with an expression vector for either GSK-3β Δ9 or an empty vector. C) The same ubiquitylation assay was performed for Nrf2 Δ17-32 -V5, Nrf2 Δ17-32,SDSGIS -V5 and Nrf2 Δ17-32,DSAPGS -V5 as in panel A, but in this case the COS1 cells were treated with 5 μM CT99021 to inhibit GSK-3.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, Ubiquitin Assay, Construct

    The Neh6 domain of Nrf2 comprises two conserved regions that include putative β-TrCP binding sites and a potential PEST sequence. A) Amino acid sequences corresponding to the Neh6 domain of Nrf2 from mouse (m), human (h), rat (r), frog (f), and zebrafish (z), along with that of ECH (i.e. chicken Nrf2) have been aligned using the T-Coffee tool (at http://www.ebi.ac.uk/Tools/msa/tcoffee/ ). White letters on a black background represent residues that are identical across at least half of the species studied, and black letters on a grey background are conserved residues. The two boxes designated SDS1 and SDS2 contain sequences enriched with Ser and Asp residues. The solid horizontal bar over residues corresponding to 347 and 385 in mouse Nrf2 depicts a potential PEST sequence that is enriched with Pro, Glu, Ser and Thr residues ( 6 ). Within the SDS1 and SDS2 boxes, a solid horizonal bar is shown above sequences that represent putative β-TrCP binding sites. The residues that are predicted to be phosphorylated by GSK-3, based on the Scansite program (at http://scansite.mit.edu ), are shown at the bottom as vertical arrows. B) Amino acid sequences of the SDS1 region in the Neh6 domain of mNrf2, hNrf2 and rNrf2 have been aligned with a similar region in the acidic domain-2 of mNrf1, hNrf1, rNrf1 and TCF11, a splice variant of Nrf1 (the protein shown is the human factor). C) Amino acid sequences of the SDS2 region in the Neh6 domain of mNrf2, hNrf2 and rNrf2 have been aligned with a similar region in the Neh6-like domain of mRn1, hNrf1, rNrf1 and TCF11.
    Figure Legend Snippet: The Neh6 domain of Nrf2 comprises two conserved regions that include putative β-TrCP binding sites and a potential PEST sequence. A) Amino acid sequences corresponding to the Neh6 domain of Nrf2 from mouse (m), human (h), rat (r), frog (f), and zebrafish (z), along with that of ECH (i.e. chicken Nrf2) have been aligned using the T-Coffee tool (at http://www.ebi.ac.uk/Tools/msa/tcoffee/ ). White letters on a black background represent residues that are identical across at least half of the species studied, and black letters on a grey background are conserved residues. The two boxes designated SDS1 and SDS2 contain sequences enriched with Ser and Asp residues. The solid horizontal bar over residues corresponding to 347 and 385 in mouse Nrf2 depicts a potential PEST sequence that is enriched with Pro, Glu, Ser and Thr residues ( 6 ). Within the SDS1 and SDS2 boxes, a solid horizonal bar is shown above sequences that represent putative β-TrCP binding sites. The residues that are predicted to be phosphorylated by GSK-3, based on the Scansite program (at http://scansite.mit.edu ), are shown at the bottom as vertical arrows. B) Amino acid sequences of the SDS1 region in the Neh6 domain of mNrf2, hNrf2 and rNrf2 have been aligned with a similar region in the acidic domain-2 of mNrf1, hNrf1, rNrf1 and TCF11, a splice variant of Nrf1 (the protein shown is the human factor). C) Amino acid sequences of the SDS2 region in the Neh6 domain of mNrf2, hNrf2 and rNrf2 have been aligned with a similar region in the Neh6-like domain of mRn1, hNrf1, rNrf1 and TCF11.

    Techniques Used: Binding Assay, Sequencing, Variant Assay

    Down-regulation of Nrf2 in human lung A549 cells decreases expression of cytoprotective genes A) A549 cells were seeded and grown in DMEM that contained 10% FBS for about 24 h before transfer to DMEM containing 0.1% FBS for 16 h. The cells were then treated for 8 h with various doses of either LY294002 or MK-2206 in DMEM containing 0.1% FBS, as indicated, before lysates were prepared and the levels of individual proteins measured by Western blotting. The antibody that recognised both phospho-GSK-3α (Ser-21) and phospho-GSK-3β (Ser-9) was from Cell Signalling (#9331). B) A549 cells were grown as described above. After serum depletion for 16 h, they were treated for 2 h with various doses of LY294002 or MK-2206 and transferred to fresh DMEM containing 0.1% FBS for a further 6 h before being harvested. Messenger RNA levels were measured by TaqMan RT-PCR.
    Figure Legend Snippet: Down-regulation of Nrf2 in human lung A549 cells decreases expression of cytoprotective genes A) A549 cells were seeded and grown in DMEM that contained 10% FBS for about 24 h before transfer to DMEM containing 0.1% FBS for 16 h. The cells were then treated for 8 h with various doses of either LY294002 or MK-2206 in DMEM containing 0.1% FBS, as indicated, before lysates were prepared and the levels of individual proteins measured by Western blotting. The antibody that recognised both phospho-GSK-3α (Ser-21) and phospho-GSK-3β (Ser-9) was from Cell Signalling (#9331). B) A549 cells were grown as described above. After serum depletion for 16 h, they were treated for 2 h with various doses of LY294002 or MK-2206 and transferred to fresh DMEM containing 0.1% FBS for a further 6 h before being harvested. Messenger RNA levels were measured by TaqMan RT-PCR.

    Techniques Used: Expressing, Western Blot, Serum Depletion, Reverse Transcription Polymerase Chain Reaction

    36) Product Images from "Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyses 1Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyses 1 [w]"

    Article Title: Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyses 1Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyses 1 [w]

    Journal: Plant Physiology

    doi: 10.1104/pp.103.025742

    Venn diagrams showing the classification of genes inducible by cold, drought, and high-salinity stresses and by ABA application identified on the basis of microarray and RNA gel-blot analyses: In total, 36 cold-inducible, 62 drought-inducible, 57 high-salinity-inducible, and 43 ABA-inducible genes were identified by cDNA microarray and confirmed by RNA gel-blot analysis. The identified genes were classified into various groups, such as cold-stress-inducible and drought-stress-inducible, genes that were up-regulated by cold, drought, and high-salinity stresses; genes that were induced by cold or drought stress and ABA application; genes that were up-regulated by cold and drought stresses; and genes that were induced by drought and high-salinity stresses. A, Intersection of genes that were up-regulated by cold stress with those that were either up-regulated by drought stress or high-salinity stress. B, Intersection of genes that were up-regulated by cold stress with those that were either up-regulated by drought stress or ABA application. C, Intersection of genes that were up-regulated by high-salinity stress with those that were either up-regulated by drought stress or ABA application.
    Figure Legend Snippet: Venn diagrams showing the classification of genes inducible by cold, drought, and high-salinity stresses and by ABA application identified on the basis of microarray and RNA gel-blot analyses: In total, 36 cold-inducible, 62 drought-inducible, 57 high-salinity-inducible, and 43 ABA-inducible genes were identified by cDNA microarray and confirmed by RNA gel-blot analysis. The identified genes were classified into various groups, such as cold-stress-inducible and drought-stress-inducible, genes that were up-regulated by cold, drought, and high-salinity stresses; genes that were induced by cold or drought stress and ABA application; genes that were up-regulated by cold and drought stresses; and genes that were induced by drought and high-salinity stresses. A, Intersection of genes that were up-regulated by cold stress with those that were either up-regulated by drought stress or high-salinity stress. B, Intersection of genes that were up-regulated by cold stress with those that were either up-regulated by drought stress or ABA application. C, Intersection of genes that were up-regulated by high-salinity stress with those that were either up-regulated by drought stress or ABA application.

    Techniques Used: Microarray, Western Blot

    37) Product Images from "Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain"

    Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-15-114

    Exon/intron structure and alternative mRNA transcripts of mouse GAD1 gene. The new arrangement of mouse GAD1 exons and introns as determined after the analysis of genomic and cDNA sequence data using bioinformatics, 3′-RACE, RT-PCR, cloning and sequencing. Exons are shown as numbered boxes (red numbers represent alternatively spliced exons) and introns as lines. Large boxes indicate the coding DNA sequence and the small boxes the 5′- and 3′-untranslated regions. The red arrowheads are showing the locations of the alternative promoters. The schematic representation of GAD1 splicing isoforms in relation to the gene is shown below the gene structure. The length in base pairs and the position of start and stop codons are indicated above each isoform. Diagrams are not to scale.
    Figure Legend Snippet: Exon/intron structure and alternative mRNA transcripts of mouse GAD1 gene. The new arrangement of mouse GAD1 exons and introns as determined after the analysis of genomic and cDNA sequence data using bioinformatics, 3′-RACE, RT-PCR, cloning and sequencing. Exons are shown as numbered boxes (red numbers represent alternatively spliced exons) and introns as lines. Large boxes indicate the coding DNA sequence and the small boxes the 5′- and 3′-untranslated regions. The red arrowheads are showing the locations of the alternative promoters. The schematic representation of GAD1 splicing isoforms in relation to the gene is shown below the gene structure. The length in base pairs and the position of start and stop codons are indicated above each isoform. Diagrams are not to scale.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Clone Assay

    Expression analysis of mouse GAD1 splicing isoforms in adult brain. (A) RT-PCR analysis of the expression of GAD1 mRNA splicing isoforms in adult mouse brain by using a forward primer either in Exon1 or Exon2 and a specific reverse primer for each transcript. (B) Analysis of the expression of Isoforms 1 to 6 by using the PCR product of lanes Exon1 and Exon2 in (A) as template for nested PCR with specific primers for each isoform. (C) Gel electrophoresis of Isoforms 3/4 and 5/6 , amplified from mouse brain cDNA using specific forward primer and reverse primer, close to the position of the polyadenylation signal. Plasmid containing Isoforms 1/2 was used as a negative control with the primers for Isoforms 3/4 and 5/6 and as a positive control with primers amplifying the 3′-end of Isoforms 1/2 . (D) Southern blotting of the gel in (C) . The membrane was probed with Probe-GAD1 against 3′-region of Isoforms 1 and 2 . Ma, marker λ/HindIII-ϕX174/HaeIII.
    Figure Legend Snippet: Expression analysis of mouse GAD1 splicing isoforms in adult brain. (A) RT-PCR analysis of the expression of GAD1 mRNA splicing isoforms in adult mouse brain by using a forward primer either in Exon1 or Exon2 and a specific reverse primer for each transcript. (B) Analysis of the expression of Isoforms 1 to 6 by using the PCR product of lanes Exon1 and Exon2 in (A) as template for nested PCR with specific primers for each isoform. (C) Gel electrophoresis of Isoforms 3/4 and 5/6 , amplified from mouse brain cDNA using specific forward primer and reverse primer, close to the position of the polyadenylation signal. Plasmid containing Isoforms 1/2 was used as a negative control with the primers for Isoforms 3/4 and 5/6 and as a positive control with primers amplifying the 3′-end of Isoforms 1/2 . (D) Southern blotting of the gel in (C) . The membrane was probed with Probe-GAD1 against 3′-region of Isoforms 1 and 2 . Ma, marker λ/HindIII-ϕX174/HaeIII.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Nested PCR, Nucleic Acid Electrophoresis, Amplification, Plasmid Preparation, Negative Control, Positive Control, Southern Blot, Marker

    Expression of GAD1 mRNA splicing isoforms and GAD2 in different mouse tissues and during development. The expression level of Isoforms 1/2 (A) , GAD2 (B) , Isoforms 3/4 (C) , Isoforms 5/6 (D) , Isoforms 7/8 (E) , Isoforms 9/10 (F) are compared by PCR amplification using mouse Multiple Tissue cDNA Panel I and cDNA from pancreas, small intestine, and large intestine as cDNA template. (G) Control PCR reaction to verify the specificity of the primers for Isoforms 1/2, 3/4 and 5/6 . In the control reaction each primer pair was tested with a plasmid containing each full length insert as a template. The template in lanes 1, 4 and 7 was plasmid containing Isoforms 1/2 as template; lanes 2, 5 and 8, plasmid containing Isoforms 5/6 ; and lanes 3, 6, 9 plasmid containing Isoforms 3/4 . Lanes (Ht) heart; (Br) brain; (Sp) spleen; (L) lung; (Li) liver; (Ms) muscle; (K) kidney; (Ts) testis; (E7) 7-day embryo; (E11) 11-day embryo; (E15) 15-day embryo; (E17) 17-day embryo; (P) pancreas; (SI) small intestine; (LI) large intestine; (N) no template control; (−) plasmid containing Isoforms 1/2 is used as template for the amplification; and Ma, marker ϕX174/HaeIII.
    Figure Legend Snippet: Expression of GAD1 mRNA splicing isoforms and GAD2 in different mouse tissues and during development. The expression level of Isoforms 1/2 (A) , GAD2 (B) , Isoforms 3/4 (C) , Isoforms 5/6 (D) , Isoforms 7/8 (E) , Isoforms 9/10 (F) are compared by PCR amplification using mouse Multiple Tissue cDNA Panel I and cDNA from pancreas, small intestine, and large intestine as cDNA template. (G) Control PCR reaction to verify the specificity of the primers for Isoforms 1/2, 3/4 and 5/6 . In the control reaction each primer pair was tested with a plasmid containing each full length insert as a template. The template in lanes 1, 4 and 7 was plasmid containing Isoforms 1/2 as template; lanes 2, 5 and 8, plasmid containing Isoforms 5/6 ; and lanes 3, 6, 9 plasmid containing Isoforms 3/4 . Lanes (Ht) heart; (Br) brain; (Sp) spleen; (L) lung; (Li) liver; (Ms) muscle; (K) kidney; (Ts) testis; (E7) 7-day embryo; (E11) 11-day embryo; (E15) 15-day embryo; (E17) 17-day embryo; (P) pancreas; (SI) small intestine; (LI) large intestine; (N) no template control; (−) plasmid containing Isoforms 1/2 is used as template for the amplification; and Ma, marker ϕX174/HaeIII.

    Techniques Used: Expressing, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Mass Spectrometry, Marker

    38) Product Images from "A Novel Gene Required for Male Fertility and Functional CATSPER Channel Formation in Spermatozoa"

    Article Title: A Novel Gene Required for Male Fertility and Functional CATSPER Channel Formation in Spermatozoa

    Journal: Nature communications

    doi: 10.1038/ncomms1153

    Molecular cloning of two alternative splice variants of CatSperδ ( a ) Tissue distribution of CatSperδ mRNA by reverse-transcription PCR. CatSperδ ( upper ) and Glyceraldehyde-3-Phosphate Dehydrogenase (control; lower ) from 12 mouse cDNAs; negative control ( lane “−” ). CatSperδ was detected only in testis. ( b and c ) Molecular cloning of CatSperδ cDNAs. Two bands differing by 118 bp were amplified by mouse testis first-strand cDNAs by PCR with primers corresponding to the most upstream 5′ sequence identified by 5′-RACE and 2 different gene-specific primers (GSP) nested at the 5′-UTR of Tmem146-s . RT, reverse transcriptase (b). Whole open reading frames (ORFs) of Tmem146 were amplified from testis cDNA (c). ( d ) Schematic diagram of CatSperδ splice variants. Two alternatively spliced mRNA variants are transcribed from the Tmem146 gene. Tmem146-s has a start site in exon 7 (blue). Tmem146-l contains a new start site in exon 1 (green) due to a change in the ORF by the additional 118 bp exon 5 (orange). The locations of probes for in situ hybridization are illustrated above the transcripts. Probe 1 is complementary to both Tmem146-s and –l . Probe 2 was amplified from Tmem146-s cDNA and corresponds to the splicing region (spanning exon 4 and 6). ( e ) Heterologous expression of CATSPERδ isoforms. V5-tagged Tmem146-s or Tmem146-l , cDNAs were transfected into HEK293T cells. After immunoprecipitation with anti-V5, immune complexes were probed with anti-V5. (f) Spatial localization of Tmem146 splice variants. Representative fields of in situ hybridization in mouse testis using antisense 1 ( upper left ) and antisense 2 ( lower left ). Sense probes served as background controls ( right panels ). Scale bar, 100 μm. ( g ) Temporal Tmem146-s and Tmem146-l mRNA levels (real time RT-PCR) during testis postnatal development. mRNAs are normalized to TATA binding protein (TBP) at each time point and expressed as ratios relative to adult (80-day) mouse testis (± SEM compared to each prior time point *P
    Figure Legend Snippet: Molecular cloning of two alternative splice variants of CatSperδ ( a ) Tissue distribution of CatSperδ mRNA by reverse-transcription PCR. CatSperδ ( upper ) and Glyceraldehyde-3-Phosphate Dehydrogenase (control; lower ) from 12 mouse cDNAs; negative control ( lane “−” ). CatSperδ was detected only in testis. ( b and c ) Molecular cloning of CatSperδ cDNAs. Two bands differing by 118 bp were amplified by mouse testis first-strand cDNAs by PCR with primers corresponding to the most upstream 5′ sequence identified by 5′-RACE and 2 different gene-specific primers (GSP) nested at the 5′-UTR of Tmem146-s . RT, reverse transcriptase (b). Whole open reading frames (ORFs) of Tmem146 were amplified from testis cDNA (c). ( d ) Schematic diagram of CatSperδ splice variants. Two alternatively spliced mRNA variants are transcribed from the Tmem146 gene. Tmem146-s has a start site in exon 7 (blue). Tmem146-l contains a new start site in exon 1 (green) due to a change in the ORF by the additional 118 bp exon 5 (orange). The locations of probes for in situ hybridization are illustrated above the transcripts. Probe 1 is complementary to both Tmem146-s and –l . Probe 2 was amplified from Tmem146-s cDNA and corresponds to the splicing region (spanning exon 4 and 6). ( e ) Heterologous expression of CATSPERδ isoforms. V5-tagged Tmem146-s or Tmem146-l , cDNAs were transfected into HEK293T cells. After immunoprecipitation with anti-V5, immune complexes were probed with anti-V5. (f) Spatial localization of Tmem146 splice variants. Representative fields of in situ hybridization in mouse testis using antisense 1 ( upper left ) and antisense 2 ( lower left ). Sense probes served as background controls ( right panels ). Scale bar, 100 μm. ( g ) Temporal Tmem146-s and Tmem146-l mRNA levels (real time RT-PCR) during testis postnatal development. mRNAs are normalized to TATA binding protein (TBP) at each time point and expressed as ratios relative to adult (80-day) mouse testis (± SEM compared to each prior time point *P

    Techniques Used: Molecular Cloning, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing, In Situ Hybridization, Expressing, Transfection, Immunoprecipitation, Quantitative RT-PCR, Binding Assay

    39) Product Images from "A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells"

    Article Title: A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054500

    Expression of T1Rs during differentiation of 3T3-L1 cells. A. The total RNAs prepared from 3T3-L1 cells at the indicated time points during differentiation were subjected to quantitative RT-PCR using mouse ribosomal protein S18 as an internal control as described in`Materials and Methods'. The mRNA levels of T1R2 and T1R3 are shown as the percentage of that of T1R3 at Day 6. Results are shown as the mean ± SE (n = 3–6). B. Immumoblot data for T1R3 and actin in undifferentiated (Day 0) and differentiated (Day 7) 3T3-L1 cells. C. Immunofluorescence staining images for T1R3 ( a and b , red) and GLUT4 ( c and d , green) in undifferentiated (Day 0, a and c ) and differentiated (Day 7, b and d ) 3T3-L1 cells. Nuclei were visualized with DAPI (blue). e , Subcellular distribution of T1R3 (red) in Day 7 cells. Arrowheads indicate peripheral localization of T1R3.
    Figure Legend Snippet: Expression of T1Rs during differentiation of 3T3-L1 cells. A. The total RNAs prepared from 3T3-L1 cells at the indicated time points during differentiation were subjected to quantitative RT-PCR using mouse ribosomal protein S18 as an internal control as described in`Materials and Methods'. The mRNA levels of T1R2 and T1R3 are shown as the percentage of that of T1R3 at Day 6. Results are shown as the mean ± SE (n = 3–6). B. Immumoblot data for T1R3 and actin in undifferentiated (Day 0) and differentiated (Day 7) 3T3-L1 cells. C. Immunofluorescence staining images for T1R3 ( a and b , red) and GLUT4 ( c and d , green) in undifferentiated (Day 0, a and c ) and differentiated (Day 7, b and d ) 3T3-L1 cells. Nuclei were visualized with DAPI (blue). e , Subcellular distribution of T1R3 (red) in Day 7 cells. Arrowheads indicate peripheral localization of T1R3.

    Techniques Used: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

    Roles for G proteins in Sweeteners Effects on Differentiation of 3T3-L1 cells. A. Expression profiles of Gαgust, Gα14 and Gαs during differentiation of 3T3-L1 cells. The total RNAs were prepared from 3T3-L1 cells as described in Fig. 1 and the mRNA levels of Gαgust, Gα14 and Gαs were measured by quantitative RT-PCR using mouse ribosomal protein S18 as an internal control. Results are shown as the mean ± SE (n = 3–6). B. 3T3-L1 cells were differentiated without (control) or with sucralose (20 mM), saccharin (20 mM), or endothelin-1 (20 nM) in the absence (0.1% DMSO) or the presence of YM-254890 (10 µM). The expression levels of PPARγ and C/EBPα at Day 2 (48 hours) were measured by immunoblotting. Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Gray and black bars show the control and the plus YM-254890 data, respectively. Results are shown as the mean values from two independent experiments. C. Undifferentiated 3T3-L1 cells were detached and transfected with the expression vectors containing wild-type or G226A mutant Gαs cDNAs (20 μg each) by electroporation as described in ‘ Materials and Methods ’. Transfected cells were seeded on a 6-well culture dish and cultured to confluence before induction of differentiation without (control) or with sucralose (20 mM) or saccharin (20 mM). The expression levels of PPARγ and C/EBPα were measured by immunoblotting at Day 2 (48 hours). Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Gray and black bars show the control and the Gαs-G226A data, respectively. Results are shown as the mean ± SE (n = 3). P
    Figure Legend Snippet: Roles for G proteins in Sweeteners Effects on Differentiation of 3T3-L1 cells. A. Expression profiles of Gαgust, Gα14 and Gαs during differentiation of 3T3-L1 cells. The total RNAs were prepared from 3T3-L1 cells as described in Fig. 1 and the mRNA levels of Gαgust, Gα14 and Gαs were measured by quantitative RT-PCR using mouse ribosomal protein S18 as an internal control. Results are shown as the mean ± SE (n = 3–6). B. 3T3-L1 cells were differentiated without (control) or with sucralose (20 mM), saccharin (20 mM), or endothelin-1 (20 nM) in the absence (0.1% DMSO) or the presence of YM-254890 (10 µM). The expression levels of PPARγ and C/EBPα at Day 2 (48 hours) were measured by immunoblotting. Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Gray and black bars show the control and the plus YM-254890 data, respectively. Results are shown as the mean values from two independent experiments. C. Undifferentiated 3T3-L1 cells were detached and transfected with the expression vectors containing wild-type or G226A mutant Gαs cDNAs (20 μg each) by electroporation as described in ‘ Materials and Methods ’. Transfected cells were seeded on a 6-well culture dish and cultured to confluence before induction of differentiation without (control) or with sucralose (20 mM) or saccharin (20 mM). The expression levels of PPARγ and C/EBPα were measured by immunoblotting at Day 2 (48 hours). Representative immunoblot data (upper panel) and the relative amounts of the proteins normalized with β-tubulin (lower panel) are shown. Gray and black bars show the control and the Gαs-G226A data, respectively. Results are shown as the mean ± SE (n = 3). P

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Mutagenesis, Electroporation, Cell Culture

    40) Product Images from "Effects of Cymbidium Root Ethanol Extract on Atopic Dermatitis"

    Article Title: Effects of Cymbidium Root Ethanol Extract on Atopic Dermatitis

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2016/5362475

    Effect of Cymbidium ethanol extract (CYM) on 2,4-dinitrochlorobenzene- (DNCB-) induced expression of interleukin- (IL-) 4, IL-13, and tumor necrosis factor- (TNF-) α mRNA in atopic dermatitis- (AD-) like mouse model. After inducing AD, 10 mg/mL CYM solution (in 3 : 1 mixture of acetone and olive oil) was applied to the dorsal skin of mice for a total of 6 times over a 2-week period. After euthanasia, dorsal skin lesions were enucleated from mice of all three groups (non-, DNCB, DNCB, and CYM treatment). Total RNA was isolated from extracted tissue of dorsal lesions using Tri-reagent. Total RNA was used as a template for cDNA synthesis, which was performed using a cDNA Synthesis kit. Real-time polymerase chain reaction (qPCR) analysis was carried out using SYBR Green I and a Lightcycler 96 instrument. The qPCR analysis was performed to detect IL-4, IL-13, and TNF- α mRNA expression. Data are mean ± standard deviation (SD, n = 6). ∗ P
    Figure Legend Snippet: Effect of Cymbidium ethanol extract (CYM) on 2,4-dinitrochlorobenzene- (DNCB-) induced expression of interleukin- (IL-) 4, IL-13, and tumor necrosis factor- (TNF-) α mRNA in atopic dermatitis- (AD-) like mouse model. After inducing AD, 10 mg/mL CYM solution (in 3 : 1 mixture of acetone and olive oil) was applied to the dorsal skin of mice for a total of 6 times over a 2-week period. After euthanasia, dorsal skin lesions were enucleated from mice of all three groups (non-, DNCB, DNCB, and CYM treatment). Total RNA was isolated from extracted tissue of dorsal lesions using Tri-reagent. Total RNA was used as a template for cDNA synthesis, which was performed using a cDNA Synthesis kit. Real-time polymerase chain reaction (qPCR) analysis was carried out using SYBR Green I and a Lightcycler 96 instrument. The qPCR analysis was performed to detect IL-4, IL-13, and TNF- α mRNA expression. Data are mean ± standard deviation (SD, n = 6). ∗ P

    Techniques Used: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

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    SYBR Green Assay:

    Article Title: Quantification of Hantaan Virus with a SYBR Green I-Based One-Step qRT-PCR Assay
    Article Snippet: .. SYBR Green Ⅰ-based one-step qRT-PCR assay qRT-PCR amplification was performed using a Bio-Rad iQ5 Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the One-Step SYBR PrimeScript RT-PCR Kit ii (TaKaRa Biotechnology, Dalian, China). .. Briefly, 2 µl of each HTNV cRNA standard was mixed with 12.5 µl of 2 × One-Step SYBR RT-PCR Buffer, 1 µl of PrimeScript One-Step Enzyme Mix 2, 1 µl of each primer (0.4 µM), and 7.5 µl of RNase-free water in 8-strip tubes (BIOplastics, Landgraaf, Netherlands).

    Article Title: Growth factors induce the improved cardiac remodeling in autologous mesenchymal stem cell-implanted failing rat hearts
    Article Snippet: .. The SYBR green real-time RT-PCR amplifications were performed using SYBR® PrimeScript™ RT-PCR Kit (TaKaRa Biotech Co.) and Applied Biosystems 7500 System (Applied Biosystems, USA). ..

    Amplification:

    Article Title: Quantification of Hantaan Virus with a SYBR Green I-Based One-Step qRT-PCR Assay
    Article Snippet: .. SYBR Green Ⅰ-based one-step qRT-PCR assay qRT-PCR amplification was performed using a Bio-Rad iQ5 Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the One-Step SYBR PrimeScript RT-PCR Kit ii (TaKaRa Biotechnology, Dalian, China). .. Briefly, 2 µl of each HTNV cRNA standard was mixed with 12.5 µl of 2 × One-Step SYBR RT-PCR Buffer, 1 µl of PrimeScript One-Step Enzyme Mix 2, 1 µl of each primer (0.4 µM), and 7.5 µl of RNase-free water in 8-strip tubes (BIOplastics, Landgraaf, Netherlands).

    Quantitative RT-PCR:

    Article Title: Quantification of Hantaan Virus with a SYBR Green I-Based One-Step qRT-PCR Assay
    Article Snippet: .. SYBR Green Ⅰ-based one-step qRT-PCR assay qRT-PCR amplification was performed using a Bio-Rad iQ5 Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the One-Step SYBR PrimeScript RT-PCR Kit ii (TaKaRa Biotechnology, Dalian, China). .. Briefly, 2 µl of each HTNV cRNA standard was mixed with 12.5 µl of 2 × One-Step SYBR RT-PCR Buffer, 1 µl of PrimeScript One-Step Enzyme Mix 2, 1 µl of each primer (0.4 µM), and 7.5 µl of RNase-free water in 8-strip tubes (BIOplastics, Landgraaf, Netherlands).

    Article Title: Growth factors induce the improved cardiac remodeling in autologous mesenchymal stem cell-implanted failing rat hearts
    Article Snippet: .. The SYBR green real-time RT-PCR amplifications were performed using SYBR® PrimeScript™ RT-PCR Kit (TaKaRa Biotech Co.) and Applied Biosystems 7500 System (Applied Biosystems, USA). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Quantification of Hantaan Virus with a SYBR Green I-Based One-Step qRT-PCR Assay
    Article Snippet: .. SYBR Green Ⅰ-based one-step qRT-PCR assay qRT-PCR amplification was performed using a Bio-Rad iQ5 Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the One-Step SYBR PrimeScript RT-PCR Kit ii (TaKaRa Biotechnology, Dalian, China). .. Briefly, 2 µl of each HTNV cRNA standard was mixed with 12.5 µl of 2 × One-Step SYBR RT-PCR Buffer, 1 µl of PrimeScript One-Step Enzyme Mix 2, 1 µl of each primer (0.4 µM), and 7.5 µl of RNase-free water in 8-strip tubes (BIOplastics, Landgraaf, Netherlands).

    Article Title: Disruption of the Sjögren-Larsson Syndrome Gene Aldh3a2 in Mice Increases Keratinocyte Growth and Retards Skin Barrier Recovery *
    Article Snippet: .. Real-time quantitative PCR was performed using the One-Step SYBR PrimeScript RT-PCR Kit II (Takara Bio) on a CFX96 Touch real-time PCR detection system (Bio-Rad) according to the manufacturer's manual. .. Forward (F) and reverse (R) primers for the respective genes were used ( ).

    Concentration Assay:

    Article Title: Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways
    Article Snippet: .. After the concentration of RNA was determined using a spectrophotometer, 1 μg of RNA was transformed into cDNA using a Prime-Script RT-PCR kit (Takara). .. The following PCR primer sequences (forward and reverse, respectively) were used: iNOS: 5′-ACA TCG ACC CGT CCA CAG TAT-3′ and 5′-CAG AGG GGT AGG CTT GTC TC-3′; COX2: 5′-ACA CAC TCT ATC ACT GGC ACC-3′ and 5′-TTC AGG GCG AAG CGT TTGC-3′; β-actin: 5′-TCT GTG TGG ATT GTG GCT CTA-3′ and 5′-CTG CTT GCT GAT CCA CAT CTG-3′.

    Article Title: Licochalcone A Upregulates Nrf2 Antioxidant Pathway and Thereby Alleviates Acetaminophen-Induced Hepatotoxicity
    Article Snippet: .. After the concentration of RNA was determined by spectrophotometer, 1 μg of RNA was transformed into cDNA using Prime-Script RT-PCR kit (Takara). .. PCR reactions were carried out using the SYBR green working solution and quantitatively measured with the Applied Biosystems 7300 real-time PCR system and software (Applied Biosystems, Carlsbad, CA, United States).

    Article Title: Lico A Enhances Nrf2-Mediated Defense Mechanisms against t-BHP-Induced Oxidative Stress and Cell Death via Akt and ERK Activation in RAW 264.7 Cells
    Article Snippet: .. After the concentration of RNA was determined by spectrophotometer, 1 μ g of RNA was transformed into cDNA using Prime-Script RT-PCR kit (Takara). .. The following PCR primer sequences (forward and reverse, resp.) were used: GCLC: 5′-ACG GCT GCT ACG ACA ACG GCC CTC-3′ and 5′-ACC CAG CGG TGC AAA CTC CGC GC-3′; GCLM: 5′-TCC TCT CGA AGA GGG CGT GTC CAG-3′ and 5′-AGG GAG G GA AGG AAG GGA GGG AG-3′; β -actin: 5′-TCT GTG TGG ATT GTG GCT CTA-3′ and 5′-CTG CTT GCT GAT CCA CAT CTG-3′.

    Spectrophotometry:

    Article Title: Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways
    Article Snippet: .. After the concentration of RNA was determined using a spectrophotometer, 1 μg of RNA was transformed into cDNA using a Prime-Script RT-PCR kit (Takara). .. The following PCR primer sequences (forward and reverse, respectively) were used: iNOS: 5′-ACA TCG ACC CGT CCA CAG TAT-3′ and 5′-CAG AGG GGT AGG CTT GTC TC-3′; COX2: 5′-ACA CAC TCT ATC ACT GGC ACC-3′ and 5′-TTC AGG GCG AAG CGT TTGC-3′; β-actin: 5′-TCT GTG TGG ATT GTG GCT CTA-3′ and 5′-CTG CTT GCT GAT CCA CAT CTG-3′.

    Article Title: Licochalcone A Upregulates Nrf2 Antioxidant Pathway and Thereby Alleviates Acetaminophen-Induced Hepatotoxicity
    Article Snippet: .. After the concentration of RNA was determined by spectrophotometer, 1 μg of RNA was transformed into cDNA using Prime-Script RT-PCR kit (Takara). .. PCR reactions were carried out using the SYBR green working solution and quantitatively measured with the Applied Biosystems 7300 real-time PCR system and software (Applied Biosystems, Carlsbad, CA, United States).

    Article Title: Lico A Enhances Nrf2-Mediated Defense Mechanisms against t-BHP-Induced Oxidative Stress and Cell Death via Akt and ERK Activation in RAW 264.7 Cells
    Article Snippet: .. After the concentration of RNA was determined by spectrophotometer, 1 μ g of RNA was transformed into cDNA using Prime-Script RT-PCR kit (Takara). .. The following PCR primer sequences (forward and reverse, resp.) were used: GCLC: 5′-ACG GCT GCT ACG ACA ACG GCC CTC-3′ and 5′-ACC CAG CGG TGC AAA CTC CGC GC-3′; GCLM: 5′-TCC TCT CGA AGA GGG CGT GTC CAG-3′ and 5′-AGG GAG G GA AGG AAG GGA GGG AG-3′; β -actin: 5′-TCT GTG TGG ATT GTG GCT CTA-3′ and 5′-CTG CTT GCT GAT CCA CAT CTG-3′.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways
    Article Snippet: .. After the concentration of RNA was determined using a spectrophotometer, 1 μg of RNA was transformed into cDNA using a Prime-Script RT-PCR kit (Takara). .. The following PCR primer sequences (forward and reverse, respectively) were used: iNOS: 5′-ACA TCG ACC CGT CCA CAG TAT-3′ and 5′-CAG AGG GGT AGG CTT GTC TC-3′; COX2: 5′-ACA CAC TCT ATC ACT GGC ACC-3′ and 5′-TTC AGG GCG AAG CGT TTGC-3′; β-actin: 5′-TCT GTG TGG ATT GTG GCT CTA-3′ and 5′-CTG CTT GCT GAT CCA CAT CTG-3′.

    Article Title: Licochalcone A Upregulates Nrf2 Antioxidant Pathway and Thereby Alleviates Acetaminophen-Induced Hepatotoxicity
    Article Snippet: .. After the concentration of RNA was determined by spectrophotometer, 1 μg of RNA was transformed into cDNA using Prime-Script RT-PCR kit (Takara). .. PCR reactions were carried out using the SYBR green working solution and quantitatively measured with the Applied Biosystems 7300 real-time PCR system and software (Applied Biosystems, Carlsbad, CA, United States).

    Article Title: Long noncoding RNA ATB promotes the epithelial−mesenchymal transition by upregulating the miR-200c/Twist1 axe and predicts poor prognosis in breast cancer
    Article Snippet: .. The RNA was reverse-transcribed into cDNA using the Prime-Script RT-PCR kit (Takara, Dalian, China) following the manufacturer’s instructions. ..

    Article Title: Effects and mechanism of aromatic aminoketone SY0916 on osteoclastic bone destruction
    Article Snippet: .. SYBR® PrimeScriptTM RT-PCR kit was from Takara. ..

    Article Title: Lico A Enhances Nrf2-Mediated Defense Mechanisms against t-BHP-Induced Oxidative Stress and Cell Death via Akt and ERK Activation in RAW 264.7 Cells
    Article Snippet: .. After the concentration of RNA was determined by spectrophotometer, 1 μ g of RNA was transformed into cDNA using Prime-Script RT-PCR kit (Takara). .. The following PCR primer sequences (forward and reverse, resp.) were used: GCLC: 5′-ACG GCT GCT ACG ACA ACG GCC CTC-3′ and 5′-ACC CAG CGG TGC AAA CTC CGC GC-3′; GCLM: 5′-TCC TCT CGA AGA GGG CGT GTC CAG-3′ and 5′-AGG GAG G GA AGG AAG GGA GGG AG-3′; β -actin: 5′-TCT GTG TGG ATT GTG GCT CTA-3′ and 5′-CTG CTT GCT GAT CCA CAT CTG-3′.

    Article Title: Quantification of Hantaan Virus with a SYBR Green I-Based One-Step qRT-PCR Assay
    Article Snippet: .. SYBR Green Ⅰ-based one-step qRT-PCR assay qRT-PCR amplification was performed using a Bio-Rad iQ5 Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with the One-Step SYBR PrimeScript RT-PCR Kit ii (TaKaRa Biotechnology, Dalian, China). .. Briefly, 2 µl of each HTNV cRNA standard was mixed with 12.5 µl of 2 × One-Step SYBR RT-PCR Buffer, 1 µl of PrimeScript One-Step Enzyme Mix 2, 1 µl of each primer (0.4 µM), and 7.5 µl of RNase-free water in 8-strip tubes (BIOplastics, Landgraaf, Netherlands).

    Article Title: Growth factors induce the improved cardiac remodeling in autologous mesenchymal stem cell-implanted failing rat hearts
    Article Snippet: .. The SYBR green real-time RT-PCR amplifications were performed using SYBR® PrimeScript™ RT-PCR Kit (TaKaRa Biotech Co.) and Applied Biosystems 7500 System (Applied Biosystems, USA). ..

    Article Title: Disruption of the Sjögren-Larsson Syndrome Gene Aldh3a2 in Mice Increases Keratinocyte Growth and Retards Skin Barrier Recovery *
    Article Snippet: .. Real-time quantitative PCR was performed using the One-Step SYBR PrimeScript RT-PCR Kit II (Takara Bio) on a CFX96 Touch real-time PCR detection system (Bio-Rad) according to the manufacturer's manual. .. Forward (F) and reverse (R) primers for the respective genes were used ( ).

    Transformation Assay:

    Article Title: Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways
    Article Snippet: .. After the concentration of RNA was determined using a spectrophotometer, 1 μg of RNA was transformed into cDNA using a Prime-Script RT-PCR kit (Takara). .. The following PCR primer sequences (forward and reverse, respectively) were used: iNOS: 5′-ACA TCG ACC CGT CCA CAG TAT-3′ and 5′-CAG AGG GGT AGG CTT GTC TC-3′; COX2: 5′-ACA CAC TCT ATC ACT GGC ACC-3′ and 5′-TTC AGG GCG AAG CGT TTGC-3′; β-actin: 5′-TCT GTG TGG ATT GTG GCT CTA-3′ and 5′-CTG CTT GCT GAT CCA CAT CTG-3′.

    Article Title: Licochalcone A Upregulates Nrf2 Antioxidant Pathway and Thereby Alleviates Acetaminophen-Induced Hepatotoxicity
    Article Snippet: .. After the concentration of RNA was determined by spectrophotometer, 1 μg of RNA was transformed into cDNA using Prime-Script RT-PCR kit (Takara). .. PCR reactions were carried out using the SYBR green working solution and quantitatively measured with the Applied Biosystems 7300 real-time PCR system and software (Applied Biosystems, Carlsbad, CA, United States).

    Article Title: Lico A Enhances Nrf2-Mediated Defense Mechanisms against t-BHP-Induced Oxidative Stress and Cell Death via Akt and ERK Activation in RAW 264.7 Cells
    Article Snippet: .. After the concentration of RNA was determined by spectrophotometer, 1 μ g of RNA was transformed into cDNA using Prime-Script RT-PCR kit (Takara). .. The following PCR primer sequences (forward and reverse, resp.) were used: GCLC: 5′-ACG GCT GCT ACG ACA ACG GCC CTC-3′ and 5′-ACC CAG CGG TGC AAA CTC CGC GC-3′; GCLM: 5′-TCC TCT CGA AGA GGG CGT GTC CAG-3′ and 5′-AGG GAG G GA AGG AAG GGA GGG AG-3′; β -actin: 5′-TCT GTG TGG ATT GTG GCT CTA-3′ and 5′-CTG CTT GCT GAT CCA CAT CTG-3′.

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  • 85
    TaKaRa mutant mouse decorin cdna
    952delT Dcn transgenic mice express both wild-type and mutant <t>decorin.</t> A: Two independent families with human CSCD have been reported. A single base pair deletion in the coding sequence at either 941 (delC) or 967 (delT) caused frameshift mutations and a truncation of the C-terminal 33 amino acids. In mice, a delG at 926 or a delT at 952 (highlighted in red) will result in a comparable frameshift mutation. The new stop codon TGA (highlighted in red) will yield a C-terminal truncated decorin comparable to that in human CSCD. B: A Cre-on approach was used to create a transgenic mouse carrying a mutant decorin <t>cDNA</t> with a deleted T at 952. A stop codon flanked with LoxP ( red triangle ) elements was inserted between a ubiquitous CAG promoter and the mutant decorin cDNA. Corneal stromal targeting of mutant decorin was generated through breeding with Kera -cre mouse. The mouse model was bred into different backgrounds, including decorin wild-type, heterozygous, and deficient backgrounds. C: Immunoblot analyses show that the transgenic mice in a decorin wild-type background expresses two decorin bands after sequential Chondroitinase ABC and PNGase F digestion, one migrating with the wild-type decorin core, the other migrating faster at ∼37 kDa, consistent with the C-terminal truncation. The level of mutant decorin was substantially less than the wild-type decorin. These qualitative immunoblots were overloaded/exposed to show the presence or absence of the truncated decorin core in mutant and wild-type corneas, respectively. The antibody used was generated against the N-terminal 17 amino acids of the decorin protein core and thus recognizes both the native and the C-terminal–truncated species. D: Breeding the mutant into a decorin-null background allowed the localization of the mutant protein core. Mutant decorin expression was identified by immunolocalization in a 952delT Dcn mouse in decorin-deficient background ( yellow arrow indicates the positive reactivity of mutant decorin in the corneal stroma). Scale bar = 25 μm.
    Mutant Mouse Decorin Cdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mutant mouse decorin cdna/product/TaKaRa
    Average 85 stars, based on 1 article reviews
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    mutant mouse decorin cdna - by Bioz Stars, 2020-09
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    98
    TaKaRa mouse brain cdna template
    Tissue distribution of the mouse and monkey UT receptor: (a) Tissue distribution of mouse UT receptor <t>cDNA</t> transcripts by RT – <t>PCR</t> revealed expression within cardiac and vascular (thoracic but not abdominal aorta) tissue in addition to bladder and pancreas. Trace levels of expression are also observed in skeletal muscle, oesophagus, lung and adipose tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (Lower panel) by Southern analysis using full-length UT receptor cDNA probe. (b) Tissue distributions of monkey UT receptor cDNA transcripts by RT – PCR revealed expression within heart (ventricle > atrium) and arterial blood vessels (aorta not vena cava), pancreas. Detectable levels of expression were also observed in the skeletal muscle, lung, thyroid and adrenal glands, kidney, upper portions of the gastrointestinal tract (oesophagus, stomach and small intestine but not colonic tissue) and spinal cord (but not in the cortical or cerebellar samples isolated). No detectable transcripts were derived from hepatic, bladder, adipose tissue or splenic tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (lower panel) by Southern analysis using full-length UT receptor cDNA probe.
    Mouse Brain Cdna Template, supplied by TaKaRa, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse brain cdna template/product/TaKaRa
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse brain cdna template - by Bioz Stars, 2020-09
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    94
    TaKaRa cdna synthesis
    Effect of Cymbidium ethanol extract (CYM) on 2,4-dinitrochlorobenzene- (DNCB-) induced expression of interleukin- (IL-) 4, IL-13, and tumor necrosis factor- (TNF-) α mRNA in atopic dermatitis- (AD-) like mouse model. After inducing AD, 10 mg/mL CYM solution (in 3 : 1 mixture of acetone and olive oil) was applied to the dorsal skin of mice for a total of 6 times over a 2-week period. After euthanasia, dorsal skin lesions were enucleated from mice of all three groups (non-, DNCB, DNCB, and CYM treatment). Total <t>RNA</t> was isolated from extracted tissue of dorsal lesions using Tri-reagent. Total RNA was used as a template for <t>cDNA</t> synthesis, which was performed using a cDNA Synthesis kit. Real-time polymerase chain reaction (qPCR) analysis was carried out using SYBR Green I and a Lightcycler 96 instrument. The qPCR analysis was performed to detect IL-4, IL-13, and TNF- α mRNA expression. Data are mean ± standard deviation (SD, n = 6). ∗ P
    Cdna Synthesis, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdna synthesis/product/TaKaRa
    Average 94 stars, based on 312 article reviews
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    cdna synthesis - by Bioz Stars, 2020-09
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    92
    TaKaRa template cdna
    Exon/intron structure and alternative mRNA transcripts of mouse GAD1 gene. The new arrangement of mouse GAD1 exons and introns as determined after the analysis of genomic and <t>cDNA</t> sequence data using bioinformatics, 3′-RACE, <t>RT-PCR,</t> cloning and sequencing. Exons are shown as numbered boxes (red numbers represent alternatively spliced exons) and introns as lines. Large boxes indicate the coding DNA sequence and the small boxes the 5′- and 3′-untranslated regions. The red arrowheads are showing the locations of the alternative promoters. The schematic representation of GAD1 splicing isoforms in relation to the gene is shown below the gene structure. The length in base pairs and the position of start and stop codons are indicated above each isoform. Diagrams are not to scale.
    Template Cdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/template cdna/product/TaKaRa
    Average 92 stars, based on 132 article reviews
    Price from $9.99 to $1999.99
    template cdna - by Bioz Stars, 2020-09
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    Image Search Results


    952delT Dcn transgenic mice express both wild-type and mutant decorin. A: Two independent families with human CSCD have been reported. A single base pair deletion in the coding sequence at either 941 (delC) or 967 (delT) caused frameshift mutations and a truncation of the C-terminal 33 amino acids. In mice, a delG at 926 or a delT at 952 (highlighted in red) will result in a comparable frameshift mutation. The new stop codon TGA (highlighted in red) will yield a C-terminal truncated decorin comparable to that in human CSCD. B: A Cre-on approach was used to create a transgenic mouse carrying a mutant decorin cDNA with a deleted T at 952. A stop codon flanked with LoxP ( red triangle ) elements was inserted between a ubiquitous CAG promoter and the mutant decorin cDNA. Corneal stromal targeting of mutant decorin was generated through breeding with Kera -cre mouse. The mouse model was bred into different backgrounds, including decorin wild-type, heterozygous, and deficient backgrounds. C: Immunoblot analyses show that the transgenic mice in a decorin wild-type background expresses two decorin bands after sequential Chondroitinase ABC and PNGase F digestion, one migrating with the wild-type decorin core, the other migrating faster at ∼37 kDa, consistent with the C-terminal truncation. The level of mutant decorin was substantially less than the wild-type decorin. These qualitative immunoblots were overloaded/exposed to show the presence or absence of the truncated decorin core in mutant and wild-type corneas, respectively. The antibody used was generated against the N-terminal 17 amino acids of the decorin protein core and thus recognizes both the native and the C-terminal–truncated species. D: Breeding the mutant into a decorin-null background allowed the localization of the mutant protein core. Mutant decorin expression was identified by immunolocalization in a 952delT Dcn mouse in decorin-deficient background ( yellow arrow indicates the positive reactivity of mutant decorin in the corneal stroma). Scale bar = 25 μm.

    Journal: The American Journal of Pathology

    Article Title: Pathophysiological Mechanisms of Autosomal Dominant Congenital Stromal Corneal Dystrophy

    doi: 10.1016/j.ajpath.2011.07.026

    Figure Lengend Snippet: 952delT Dcn transgenic mice express both wild-type and mutant decorin. A: Two independent families with human CSCD have been reported. A single base pair deletion in the coding sequence at either 941 (delC) or 967 (delT) caused frameshift mutations and a truncation of the C-terminal 33 amino acids. In mice, a delG at 926 or a delT at 952 (highlighted in red) will result in a comparable frameshift mutation. The new stop codon TGA (highlighted in red) will yield a C-terminal truncated decorin comparable to that in human CSCD. B: A Cre-on approach was used to create a transgenic mouse carrying a mutant decorin cDNA with a deleted T at 952. A stop codon flanked with LoxP ( red triangle ) elements was inserted between a ubiquitous CAG promoter and the mutant decorin cDNA. Corneal stromal targeting of mutant decorin was generated through breeding with Kera -cre mouse. The mouse model was bred into different backgrounds, including decorin wild-type, heterozygous, and deficient backgrounds. C: Immunoblot analyses show that the transgenic mice in a decorin wild-type background expresses two decorin bands after sequential Chondroitinase ABC and PNGase F digestion, one migrating with the wild-type decorin core, the other migrating faster at ∼37 kDa, consistent with the C-terminal truncation. The level of mutant decorin was substantially less than the wild-type decorin. These qualitative immunoblots were overloaded/exposed to show the presence or absence of the truncated decorin core in mutant and wild-type corneas, respectively. The antibody used was generated against the N-terminal 17 amino acids of the decorin protein core and thus recognizes both the native and the C-terminal–truncated species. D: Breeding the mutant into a decorin-null background allowed the localization of the mutant protein core. Mutant decorin expression was identified by immunolocalization in a 952delT Dcn mouse in decorin-deficient background ( yellow arrow indicates the positive reactivity of mutant decorin in the corneal stroma). Scale bar = 25 μm.

    Article Snippet: A pMSCV/Dec plasmid, which contains pMSCVpuro (Clontech, Mountain View CA) vector, and a full-length mouse decorin cDNA, was used as the template for making the mutant mouse decorin cDNA (952 delT) with the use of a Transformer Site-directed mutagenesis kit (Clontech).

    Techniques: Transgenic Assay, Mouse Assay, Mutagenesis, Sequencing, Generated, Western Blot, Expressing

    Tissue distribution of the mouse and monkey UT receptor: (a) Tissue distribution of mouse UT receptor cDNA transcripts by RT – PCR revealed expression within cardiac and vascular (thoracic but not abdominal aorta) tissue in addition to bladder and pancreas. Trace levels of expression are also observed in skeletal muscle, oesophagus, lung and adipose tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (Lower panel) by Southern analysis using full-length UT receptor cDNA probe. (b) Tissue distributions of monkey UT receptor cDNA transcripts by RT – PCR revealed expression within heart (ventricle > atrium) and arterial blood vessels (aorta not vena cava), pancreas. Detectable levels of expression were also observed in the skeletal muscle, lung, thyroid and adrenal glands, kidney, upper portions of the gastrointestinal tract (oesophagus, stomach and small intestine but not colonic tissue) and spinal cord (but not in the cortical or cerebellar samples isolated). No detectable transcripts were derived from hepatic, bladder, adipose tissue or splenic tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (lower panel) by Southern analysis using full-length UT receptor cDNA probe.

    Journal: British Journal of Pharmacology

    Article Title: Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey

    doi: 10.1038/sj.bjp.0704671

    Figure Lengend Snippet: Tissue distribution of the mouse and monkey UT receptor: (a) Tissue distribution of mouse UT receptor cDNA transcripts by RT – PCR revealed expression within cardiac and vascular (thoracic but not abdominal aorta) tissue in addition to bladder and pancreas. Trace levels of expression are also observed in skeletal muscle, oesophagus, lung and adipose tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (Lower panel) by Southern analysis using full-length UT receptor cDNA probe. (b) Tissue distributions of monkey UT receptor cDNA transcripts by RT – PCR revealed expression within heart (ventricle > atrium) and arterial blood vessels (aorta not vena cava), pancreas. Detectable levels of expression were also observed in the skeletal muscle, lung, thyroid and adrenal glands, kidney, upper portions of the gastrointestinal tract (oesophagus, stomach and small intestine but not colonic tissue) and spinal cord (but not in the cortical or cerebellar samples isolated). No detectable transcripts were derived from hepatic, bladder, adipose tissue or splenic tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (lower panel) by Southern analysis using full-length UT receptor cDNA probe.

    Article Snippet: These primers were used to obtain the full-length preproU-II complementary DNA (cDNA) clone from mouse brain cDNA template (Clonetech) by polymerase chain reaction (PCR).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Isolation, Derivative Assay

    Tissue distribution of the mouse and monkey U-II. (a): Tissue distribution of mouse preproU-II cDNA transcripts by RT – PCR revealed expression within heart, thoracic aorta, testes, brain, skeletal muscle, liver, kidney and spleen (upper panel). Negligible expression of preproU-II was observed in the mouse gastrointestinal tract (stomach, oesophagus, small intestine and colon), bladder, pancreas, adrenal, lung and adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel). (b) Tissue distribution of monkey preproU-II cDNA transcripts by RT – PCR revealed expression within heart (ventricle and atrium), thoracic aorta, CNS (spinal cord, cerebellum and cortex), skeletal muscle, kidney, liver and spleen (upper panel). No detectable transcripts were derived from vena cava, endocrine tissues including thyroid, pancreas and adrenal glands, lung, gastrointestinal tissue (oesophagus, stomach, small intestine, colon), bladder or adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel).

    Journal: British Journal of Pharmacology

    Article Title: Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey

    doi: 10.1038/sj.bjp.0704671

    Figure Lengend Snippet: Tissue distribution of the mouse and monkey U-II. (a): Tissue distribution of mouse preproU-II cDNA transcripts by RT – PCR revealed expression within heart, thoracic aorta, testes, brain, skeletal muscle, liver, kidney and spleen (upper panel). Negligible expression of preproU-II was observed in the mouse gastrointestinal tract (stomach, oesophagus, small intestine and colon), bladder, pancreas, adrenal, lung and adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel). (b) Tissue distribution of monkey preproU-II cDNA transcripts by RT – PCR revealed expression within heart (ventricle and atrium), thoracic aorta, CNS (spinal cord, cerebellum and cortex), skeletal muscle, kidney, liver and spleen (upper panel). No detectable transcripts were derived from vena cava, endocrine tissues including thyroid, pancreas and adrenal glands, lung, gastrointestinal tissue (oesophagus, stomach, small intestine, colon), bladder or adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel).

    Article Snippet: These primers were used to obtain the full-length preproU-II complementary DNA (cDNA) clone from mouse brain cDNA template (Clonetech) by polymerase chain reaction (PCR).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Derivative Assay

    Effect of Cymbidium ethanol extract (CYM) on 2,4-dinitrochlorobenzene- (DNCB-) induced expression of interleukin- (IL-) 4, IL-13, and tumor necrosis factor- (TNF-) α mRNA in atopic dermatitis- (AD-) like mouse model. After inducing AD, 10 mg/mL CYM solution (in 3 : 1 mixture of acetone and olive oil) was applied to the dorsal skin of mice for a total of 6 times over a 2-week period. After euthanasia, dorsal skin lesions were enucleated from mice of all three groups (non-, DNCB, DNCB, and CYM treatment). Total RNA was isolated from extracted tissue of dorsal lesions using Tri-reagent. Total RNA was used as a template for cDNA synthesis, which was performed using a cDNA Synthesis kit. Real-time polymerase chain reaction (qPCR) analysis was carried out using SYBR Green I and a Lightcycler 96 instrument. The qPCR analysis was performed to detect IL-4, IL-13, and TNF- α mRNA expression. Data are mean ± standard deviation (SD, n = 6). ∗ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Effects of Cymbidium Root Ethanol Extract on Atopic Dermatitis

    doi: 10.1155/2016/5362475

    Figure Lengend Snippet: Effect of Cymbidium ethanol extract (CYM) on 2,4-dinitrochlorobenzene- (DNCB-) induced expression of interleukin- (IL-) 4, IL-13, and tumor necrosis factor- (TNF-) α mRNA in atopic dermatitis- (AD-) like mouse model. After inducing AD, 10 mg/mL CYM solution (in 3 : 1 mixture of acetone and olive oil) was applied to the dorsal skin of mice for a total of 6 times over a 2-week period. After euthanasia, dorsal skin lesions were enucleated from mice of all three groups (non-, DNCB, DNCB, and CYM treatment). Total RNA was isolated from extracted tissue of dorsal lesions using Tri-reagent. Total RNA was used as a template for cDNA synthesis, which was performed using a cDNA Synthesis kit. Real-time polymerase chain reaction (qPCR) analysis was carried out using SYBR Green I and a Lightcycler 96 instrument. The qPCR analysis was performed to detect IL-4, IL-13, and TNF- α mRNA expression. Data are mean ± standard deviation (SD, n = 6). ∗ P

    Article Snippet: Total RNA was used as a template for cDNA synthesis, which was performed using a cDNA Synthesis kit (Takara Bio, Shiga, Japan).

    Techniques: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    Exon/intron structure and alternative mRNA transcripts of mouse GAD1 gene. The new arrangement of mouse GAD1 exons and introns as determined after the analysis of genomic and cDNA sequence data using bioinformatics, 3′-RACE, RT-PCR, cloning and sequencing. Exons are shown as numbered boxes (red numbers represent alternatively spliced exons) and introns as lines. Large boxes indicate the coding DNA sequence and the small boxes the 5′- and 3′-untranslated regions. The red arrowheads are showing the locations of the alternative promoters. The schematic representation of GAD1 splicing isoforms in relation to the gene is shown below the gene structure. The length in base pairs and the position of start and stop codons are indicated above each isoform. Diagrams are not to scale.

    Journal: BMC Neuroscience

    Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

    doi: 10.1186/1471-2202-15-114

    Figure Lengend Snippet: Exon/intron structure and alternative mRNA transcripts of mouse GAD1 gene. The new arrangement of mouse GAD1 exons and introns as determined after the analysis of genomic and cDNA sequence data using bioinformatics, 3′-RACE, RT-PCR, cloning and sequencing. Exons are shown as numbered boxes (red numbers represent alternatively spliced exons) and introns as lines. Large boxes indicate the coding DNA sequence and the small boxes the 5′- and 3′-untranslated regions. The red arrowheads are showing the locations of the alternative promoters. The schematic representation of GAD1 splicing isoforms in relation to the gene is shown below the gene structure. The length in base pairs and the position of start and stop codons are indicated above each isoform. Diagrams are not to scale.

    Article Snippet: The PCR mix (25 μl) contained 2.5 μl template cDNA, 1 μM forward and reverse primers, 200 μM dNTPs (Takara), and 0.5 U Ex Taq HS (Takara).

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Clone Assay

    Expression analysis of mouse GAD1 splicing isoforms in adult brain. (A) RT-PCR analysis of the expression of GAD1 mRNA splicing isoforms in adult mouse brain by using a forward primer either in Exon1 or Exon2 and a specific reverse primer for each transcript. (B) Analysis of the expression of Isoforms 1 to 6 by using the PCR product of lanes Exon1 and Exon2 in (A) as template for nested PCR with specific primers for each isoform. (C) Gel electrophoresis of Isoforms 3/4 and 5/6 , amplified from mouse brain cDNA using specific forward primer and reverse primer, close to the position of the polyadenylation signal. Plasmid containing Isoforms 1/2 was used as a negative control with the primers for Isoforms 3/4 and 5/6 and as a positive control with primers amplifying the 3′-end of Isoforms 1/2 . (D) Southern blotting of the gel in (C) . The membrane was probed with Probe-GAD1 against 3′-region of Isoforms 1 and 2 . Ma, marker λ/HindIII-ϕX174/HaeIII.

    Journal: BMC Neuroscience

    Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

    doi: 10.1186/1471-2202-15-114

    Figure Lengend Snippet: Expression analysis of mouse GAD1 splicing isoforms in adult brain. (A) RT-PCR analysis of the expression of GAD1 mRNA splicing isoforms in adult mouse brain by using a forward primer either in Exon1 or Exon2 and a specific reverse primer for each transcript. (B) Analysis of the expression of Isoforms 1 to 6 by using the PCR product of lanes Exon1 and Exon2 in (A) as template for nested PCR with specific primers for each isoform. (C) Gel electrophoresis of Isoforms 3/4 and 5/6 , amplified from mouse brain cDNA using specific forward primer and reverse primer, close to the position of the polyadenylation signal. Plasmid containing Isoforms 1/2 was used as a negative control with the primers for Isoforms 3/4 and 5/6 and as a positive control with primers amplifying the 3′-end of Isoforms 1/2 . (D) Southern blotting of the gel in (C) . The membrane was probed with Probe-GAD1 against 3′-region of Isoforms 1 and 2 . Ma, marker λ/HindIII-ϕX174/HaeIII.

    Article Snippet: The PCR mix (25 μl) contained 2.5 μl template cDNA, 1 μM forward and reverse primers, 200 μM dNTPs (Takara), and 0.5 U Ex Taq HS (Takara).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Nested PCR, Nucleic Acid Electrophoresis, Amplification, Plasmid Preparation, Negative Control, Positive Control, Southern Blot, Marker

    Expression of GAD1 mRNA splicing isoforms and GAD2 in different mouse tissues and during development. The expression level of Isoforms 1/2 (A) , GAD2 (B) , Isoforms 3/4 (C) , Isoforms 5/6 (D) , Isoforms 7/8 (E) , Isoforms 9/10 (F) are compared by PCR amplification using mouse Multiple Tissue cDNA Panel I and cDNA from pancreas, small intestine, and large intestine as cDNA template. (G) Control PCR reaction to verify the specificity of the primers for Isoforms 1/2, 3/4 and 5/6 . In the control reaction each primer pair was tested with a plasmid containing each full length insert as a template. The template in lanes 1, 4 and 7 was plasmid containing Isoforms 1/2 as template; lanes 2, 5 and 8, plasmid containing Isoforms 5/6 ; and lanes 3, 6, 9 plasmid containing Isoforms 3/4 . Lanes (Ht) heart; (Br) brain; (Sp) spleen; (L) lung; (Li) liver; (Ms) muscle; (K) kidney; (Ts) testis; (E7) 7-day embryo; (E11) 11-day embryo; (E15) 15-day embryo; (E17) 17-day embryo; (P) pancreas; (SI) small intestine; (LI) large intestine; (N) no template control; (−) plasmid containing Isoforms 1/2 is used as template for the amplification; and Ma, marker ϕX174/HaeIII.

    Journal: BMC Neuroscience

    Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

    doi: 10.1186/1471-2202-15-114

    Figure Lengend Snippet: Expression of GAD1 mRNA splicing isoforms and GAD2 in different mouse tissues and during development. The expression level of Isoforms 1/2 (A) , GAD2 (B) , Isoforms 3/4 (C) , Isoforms 5/6 (D) , Isoforms 7/8 (E) , Isoforms 9/10 (F) are compared by PCR amplification using mouse Multiple Tissue cDNA Panel I and cDNA from pancreas, small intestine, and large intestine as cDNA template. (G) Control PCR reaction to verify the specificity of the primers for Isoforms 1/2, 3/4 and 5/6 . In the control reaction each primer pair was tested with a plasmid containing each full length insert as a template. The template in lanes 1, 4 and 7 was plasmid containing Isoforms 1/2 as template; lanes 2, 5 and 8, plasmid containing Isoforms 5/6 ; and lanes 3, 6, 9 plasmid containing Isoforms 3/4 . Lanes (Ht) heart; (Br) brain; (Sp) spleen; (L) lung; (Li) liver; (Ms) muscle; (K) kidney; (Ts) testis; (E7) 7-day embryo; (E11) 11-day embryo; (E15) 15-day embryo; (E17) 17-day embryo; (P) pancreas; (SI) small intestine; (LI) large intestine; (N) no template control; (−) plasmid containing Isoforms 1/2 is used as template for the amplification; and Ma, marker ϕX174/HaeIII.

    Article Snippet: The PCR mix (25 μl) contained 2.5 μl template cDNA, 1 μM forward and reverse primers, 200 μM dNTPs (Takara), and 0.5 U Ex Taq HS (Takara).

    Techniques: Expressing, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Mass Spectrometry, Marker