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Santa Cruz Biotechnology mouse top1 sirna

L1-70 interacts with <t>TOP1,</t> PPARγ and NDUFV2, but not with other L1 binding partners in neurons. Cultured cortical neurons were treated with vehicle (-) or with the serine protease inhibitor aprotinin. They were then subjected to proximity ligation with L1 antibody and antibodies against SFPQ, NonO, PSPC1, WDR5, TOP1, HistH1, Nup93, Hsc70, SYT1, impβ1, ERα, RXR, PPARγ, NDUFV2, AR, VDR or GAPDH. ( a ) Representative images of vehicle- and aprotinin-treated neurons stained with the mouse L1 antibody C-2 and rabbit antibodies against TOP1, PPARγ or NDUFV2 are shown. Nuclei are stained with DAPI. Scale bars: 10 µm. ( b ) Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of vehicle control set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). ( c ) Cortical neurons were treated with or without L1 antibody 557. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatment without antibody 557 set to 100%). (*** p < 0.005; one-way ANOVA with Bonferroni’s multiple comparison test). ( d ) Cortical and cerebellar neurons pretreated with vehicle or aprotinin were incubated without or with antibody 557 in the absence or presence of aprotinin. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatments without aprotinin set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test).

Mouse Top1 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions"

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms24032097

L1-70 interacts with TOP1, PPARγ and NDUFV2, but not with other L1 binding partners in neurons. Cultured cortical neurons were treated with vehicle (-) or with the serine protease inhibitor aprotinin. They were then subjected to proximity ligation with L1 antibody and antibodies against SFPQ, NonO, PSPC1, WDR5, TOP1, HistH1, Nup93, Hsc70, SYT1, impβ1, ERα, RXR, PPARγ, NDUFV2, AR, VDR or GAPDH. ( a ) Representative images of vehicle- and aprotinin-treated neurons stained with the mouse L1 antibody C-2 and rabbit antibodies against TOP1, PPARγ or NDUFV2 are shown. Nuclei are stained with DAPI. Scale bars: 10 µm. ( b ) Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of vehicle control set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). ( c ) Cortical neurons were treated with or without L1 antibody 557. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatment without antibody 557 set to 100%). (*** p < 0.005; one-way ANOVA with Bonferroni’s multiple comparison test). ( d ) Cortical and cerebellar neurons pretreated with vehicle or aprotinin were incubated without or with antibody 557 in the absence or presence of aprotinin. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatments without aprotinin set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test).

Figure Legend Snippet: L1-70 interacts with TOP1, PPARγ and NDUFV2, but not with other L1 binding partners in neurons. Cultured cortical neurons were treated with vehicle (-) or with the serine protease inhibitor aprotinin. They were then subjected to proximity ligation with L1 antibody and antibodies against SFPQ, NonO, PSPC1, WDR5, TOP1, HistH1, Nup93, Hsc70, SYT1, impβ1, ERα, RXR, PPARγ, NDUFV2, AR, VDR or GAPDH. ( a ) Representative images of vehicle- and aprotinin-treated neurons stained with the mouse L1 antibody C-2 and rabbit antibodies against TOP1, PPARγ or NDUFV2 are shown. Nuclei are stained with DAPI. Scale bars: 10 µm. ( b ) Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of vehicle control set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). ( c ) Cortical neurons were treated with or without L1 antibody 557. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatment without antibody 557 set to 100%). (*** p < 0.005; one-way ANOVA with Bonferroni’s multiple comparison test). ( d ) Cortical and cerebellar neurons pretreated with vehicle or aprotinin were incubated without or with antibody 557 in the absence or presence of aprotinin. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatments without aprotinin set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test).

Techniques Used: Binding Assay, Cell Culture, Protease Inhibitor, Ligation, Staining, Control, Comparison, Incubation

L1-ICD binds to TOP1 and PPARγ. Recombinant TOP1 ( a ) and PPARγ ( b ) were substrate-coated and incubated with increasing concentrations of L1-ICD. Binding of L1-ICD was determined by ELISA using mouse L1 antibody C-2 and horseradish peroxidase-conjugated secondary antibody. Mean values ± SD from three independent experiments carried out in triplicate are shown. ( c ) Recombinant TOP1 and PPARγ were substrate-coated and incubated with L1-ICD in the absence or presence of a five-fold excess of L1 peptides P1, P2, P3, P4 or P5, which cover the entire L1-ICD sequence. The ICD and transmembrane domain (TMD) of L1 and the positions of the peptides in L1-ICD are visualized. Binding was determined by ELISA using mouse L1 antibody C-2 and horseradish peroxidase-conjugated secondary antibody. Mean values + SD from five independent experiments carried out in triplicate are shown for the binding relative to control (values in the absence of peptides set to 100%) (** p < 0.01, *** p < 0.005, **** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test).

Figure Legend Snippet: L1-ICD binds to TOP1 and PPARγ. Recombinant TOP1 ( a ) and PPARγ ( b ) were substrate-coated and incubated with increasing concentrations of L1-ICD. Binding of L1-ICD was determined by ELISA using mouse L1 antibody C-2 and horseradish peroxidase-conjugated secondary antibody. Mean values ± SD from three independent experiments carried out in triplicate are shown. ( c ) Recombinant TOP1 and PPARγ were substrate-coated and incubated with L1-ICD in the absence or presence of a five-fold excess of L1 peptides P1, P2, P3, P4 or P5, which cover the entire L1-ICD sequence. The ICD and transmembrane domain (TMD) of L1 and the positions of the peptides in L1-ICD are visualized. Binding was determined by ELISA using mouse L1 antibody C-2 and horseradish peroxidase-conjugated secondary antibody. Mean values + SD from five independent experiments carried out in triplicate are shown for the binding relative to control (values in the absence of peptides set to 100%) (** p < 0.01, *** p < 0.005, **** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test).

Techniques Used: Recombinant, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Control, Comparison

$No enhanced interaction of L1 with TOP1, PPARγ and NDUFV2 in stimulated cortical neurons from L1-70-lacking mutant mice. Cultured cortical neurons from L1/687 ( a ) and L1/858–863 ( b ) mutant mice and their wild-type littermates ( a , b ) were treated without (-) or with L1 antibody 557 (557) and then subjected to proximity ligation assay with L1 antibody and TOP1, PPARγ or NDUFV2 antibodies. ( a ) For control, the L1 antibody and an antibody against SFPQ, ERα or RXR were used for proximity ligation. ( a , b ) Mean values + SD are shown for the average numbers of L1/TOP1-, L1/NDUFV2-, L1/PPARγ-, L1/SFPQ-, L1/ERα-, and L1/RXR-positive dots per cell relative to control (values of unstimulated wild-type neurons set to 100%) (** p < 0.01, **** p < 0.001 relative to non-stimulated wild-type neurons, ## p < 0.01, #### p < 0.001 relative to stimulated wild-type neurons; one-way ANOVA with Dunn’s multiple comparison test). ( c ) Non-nuclear brain fractions from wild-type (WT) and L1/858–863 mutant (Mut) mice were subjected to immunoprecipitation (IP) with immobilized mouse antibodies against TOP1, PPARγ and NDUFV2. Non-nuclear fractions (input) and immunoprecipitates were subjected to Western blot (WB) analysis with L1 antibody C-2. The arrowhead indicates L1-70.$
Figure Legend Snippet: No enhanced interaction of L1 with TOP1, PPARγ and NDUFV2 in stimulated cortical neurons from L1-70-lacking mutant mice. Cultured cortical neurons from L1/687 ( a ) and L1/858–863 ( b ) mutant mice and their wild-type littermates ( a , b ) were treated without (-) or with L1 antibody 557 (557) and then subjected to proximity ligation assay with L1 antibody and TOP1, PPARγ or NDUFV2 antibodies. ( a ) For control, the L1 antibody and an antibody against SFPQ, ERα or RXR were used for proximity ligation. ( a , b ) Mean values + SD are shown for the average numbers of L1/TOP1-, L1/NDUFV2-, L1/PPARγ-, L1/SFPQ-, L1/ERα-, and L1/RXR-positive dots per cell relative to control (values of unstimulated wild-type neurons set to 100%) (** p < 0.01, **** p < 0.001 relative to non-stimulated wild-type neurons, ## p < 0.01, #### p < 0.001 relative to stimulated wild-type neurons; one-way ANOVA with Dunn’s multiple comparison test). ( c ) Non-nuclear brain fractions from wild-type (WT) and L1/858–863 mutant (Mut) mice were subjected to immunoprecipitation (IP) with immobilized mouse antibodies against TOP1, PPARγ and NDUFV2. Non-nuclear fractions (input) and immunoprecipitates were subjected to Western blot (WB) analysis with L1 antibody C-2. The arrowhead indicates L1-70.

Techniques Used: Mutagenesis, Cell Culture, Proximity Ligation Assay, Control, Ligation, Comparison, Immunoprecipitation, Western Blot

Reduction of TOP1, PPARγ or NDUFV2 expression inhibits L1-dependent neurite outgrowth. ( a , b ) Cortical neurons were mock-transfected or transfected with control siRNA, TOP1 siRNA, PPARγ siRNA or NDUFV2 siRNA. Neurons were then treated without or with L1 antibody 557. ( a , b ) Mean values + SEM from three independent experiments are shown for total neurite lengths (**** p < 0.001 relative to mock-transfected non-stimulated neurons, ### p < 0.005, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Dunn’s multiple comparison test).

Figure Legend Snippet: Reduction of TOP1, PPARγ or NDUFV2 expression inhibits L1-dependent neurite outgrowth. ( a , b ) Cortical neurons were mock-transfected or transfected with control siRNA, TOP1 siRNA, PPARγ siRNA or NDUFV2 siRNA. Neurons were then treated without or with L1 antibody 557. ( a , b ) Mean values + SEM from three independent experiments are shown for total neurite lengths (**** p < 0.001 relative to mock-transfected non-stimulated neurons, ### p < 0.005, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Dunn’s multiple comparison test).

Techniques Used: Expressing, Transfection, Control, Comparison

The interaction of L1 with TOP1, PPARγ or NDUFV2 is reduced by L1, TOP1, PPARγ and NDUFV2 siRNAs. ( a ) Cultured cortical neurons were mock-transfected or transfected with TOP1, PPARγ, NDUFV2 or L1 siRNAs. The transfected cells were then subjected to proximity ligation with L1 antibody and antibodies against TOP1, PPARγ or NDUFV2. Mean values + SD from two experiments are shown for the average numbers of L1/TOP1-, L1/PPARγ- and L1/NDUFV2-positive red dots per cell relative to control (values of mock-transfected neurons set to 100%) (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). ( b ) Western blot analysis of lysates from mock-transfected neurons or neurons transfected with TOP1 siRNA using TOP1 antibody. Ponceau S staining of a prominent 35 kDa band served as loading control. ( c ) Mock-transfected neurons or neurons transfected with PPARγ or NDUFV2 siRNAs were subjected to immunostaining with PPARγ or NDUFV2 antibody. Nuclei are stained with DAPI. Representative images are shown and indicate reduced PPARγ or NDUFV2 levels in neurons transfected with PPARγ siRNA or NDUFV2 siRNA. Scale bars: 10 µm.

Figure Legend Snippet: The interaction of L1 with TOP1, PPARγ or NDUFV2 is reduced by L1, TOP1, PPARγ and NDUFV2 siRNAs. ( a ) Cultured cortical neurons were mock-transfected or transfected with TOP1, PPARγ, NDUFV2 or L1 siRNAs. The transfected cells were then subjected to proximity ligation with L1 antibody and antibodies against TOP1, PPARγ or NDUFV2. Mean values + SD from two experiments are shown for the average numbers of L1/TOP1-, L1/PPARγ- and L1/NDUFV2-positive red dots per cell relative to control (values of mock-transfected neurons set to 100%) (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). ( b ) Western blot analysis of lysates from mock-transfected neurons or neurons transfected with TOP1 siRNA using TOP1 antibody. Ponceau S staining of a prominent 35 kDa band served as loading control. ( c ) Mock-transfected neurons or neurons transfected with PPARγ or NDUFV2 siRNAs were subjected to immunostaining with PPARγ or NDUFV2 antibody. Nuclei are stained with DAPI. Representative images are shown and indicate reduced PPARγ or NDUFV2 levels in neurons transfected with PPARγ siRNA or NDUFV2 siRNA. Scale bars: 10 µm.

Techniques Used: Cell Culture, Transfection, Ligation, Control, Comparison, Western Blot, Staining, Immunostaining

Disturbance of the L1/TOP1 and L1/PPARγ interactions inhibits L1-dependent neurite outgrowth from cortical neurons. ( a , b ) Cortical neurons were treated without (-) or with tat-P4 peptide. Neurons were then treated without or with L1 antibody 557 (557). ( a ) Mean values + SEM from three independent experiments are shown for total neurite lengths (**** p < 0.001 relative to mock-transfected non-stimulated neurons, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Dunn’s multiple comparison test). ( b ) After the treatments, neurons were subjected to proximity ligation with L1 antibody and TOP1 antibody or PPARγ antibody. Mean values + SD from three independent experiments are shown for average numbers of L1/TOP1- and L1/PPARγ-positive red dots per cell relative to control (values of non-transfected neurons set to 100%) (*** p < 0.005, **** p < 0.001 relative to mock-transfected stimulated neurons, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Bonferroni’s multiple comparison test).

Figure Legend Snippet: Disturbance of the L1/TOP1 and L1/PPARγ interactions inhibits L1-dependent neurite outgrowth from cortical neurons. ( a , b ) Cortical neurons were treated without (-) or with tat-P4 peptide. Neurons were then treated without or with L1 antibody 557 (557). ( a ) Mean values + SEM from three independent experiments are shown for total neurite lengths (**** p < 0.001 relative to mock-transfected non-stimulated neurons, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Dunn’s multiple comparison test). ( b ) After the treatments, neurons were subjected to proximity ligation with L1 antibody and TOP1 antibody or PPARγ antibody. Mean values + SD from three independent experiments are shown for average numbers of L1/TOP1- and L1/PPARγ-positive red dots per cell relative to control (values of non-transfected neurons set to 100%) (*** p < 0.005, **** p < 0.001 relative to mock-transfected stimulated neurons, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Bonferroni’s multiple comparison test).

Techniques Used: Transfection, Comparison, Ligation, Control

Topoisomerase inhibitors reduce L1-dependent neurite outgrowth as well as neuronal survival and migration. Cerebellar neurons ( a , c ) and cerebellar explants ( e ), and cortical neurons ( b , d ) were treated without (-) or with topotecan (Topo) or irinotecan (Irino) in the absence or presence of L1 antibody 557. ( a , b ) Mean values + SEM from three independent experiments are shown for total neurite lengths (*** p < 0.005, **** p < 0.001 relative to non-stimulated neurons in the absence of inhibitors, ### p < 0.005, #### p < 0.001 relative to stimulated neurons in the absence of inhibitors; one-way ANOVA with Dunn’s multiple comparison test). ( c , d ) Mean values + SEM from three independent experiments are shown for the numbers of viable cells relative to control (values of unstimulated neurons in the absence of H 2 O 2 set to 100%) (#### p < 0.0001 relative to stimulated neurons in absence of inhibitors and the presence of H 2 O 2 , **** p < 0.0001 relative to non-stimulated neurons in absence of inhibitors and the presence of H 2 O 2 , §§§§ p < 0.0001 relative to non-stimulated neurons in absence of inhibitors and H 2 O 2 ; one-way ANOVA with Dunn’s multiple comparison test). ( e ) Mean values + SEM from three independent experiments are shown for the total numbers of migrating cells (### p < 0.005 relative to stimulated neurons in absence of inhibitors, *** p < 0.005 relative to non-stimulated neurons in absence of inhibitors; one-way ANOVA with Dunn’s multiple comparison test). ( f ) Nuclear extracts from cortical neurons transfected without (mock) or with control siRNA, L1 siRNA or TOP1 siRNA were incubated with supercoiled pUC19 plasmid DNA. For control, the vector was incubated with the reaction buffer. The samples were then subjected to agarose gel electrophoresis and ethidium bromide staining. A representative image of a stained gel is shown.

Figure Legend Snippet: Topoisomerase inhibitors reduce L1-dependent neurite outgrowth as well as neuronal survival and migration. Cerebellar neurons ( a , c ) and cerebellar explants ( e ), and cortical neurons ( b , d ) were treated without (-) or with topotecan (Topo) or irinotecan (Irino) in the absence or presence of L1 antibody 557. ( a , b ) Mean values + SEM from three independent experiments are shown for total neurite lengths (*** p < 0.005, **** p < 0.001 relative to non-stimulated neurons in the absence of inhibitors, ### p < 0.005, #### p < 0.001 relative to stimulated neurons in the absence of inhibitors; one-way ANOVA with Dunn’s multiple comparison test). ( c , d ) Mean values + SEM from three independent experiments are shown for the numbers of viable cells relative to control (values of unstimulated neurons in the absence of H 2 O 2 set to 100%) (#### p < 0.0001 relative to stimulated neurons in absence of inhibitors and the presence of H 2 O 2 , **** p < 0.0001 relative to non-stimulated neurons in absence of inhibitors and the presence of H 2 O 2 , §§§§ p < 0.0001 relative to non-stimulated neurons in absence of inhibitors and H 2 O 2 ; one-way ANOVA with Dunn’s multiple comparison test). ( e ) Mean values + SEM from three independent experiments are shown for the total numbers of migrating cells (### p < 0.005 relative to stimulated neurons in absence of inhibitors, *** p < 0.005 relative to non-stimulated neurons in absence of inhibitors; one-way ANOVA with Dunn’s multiple comparison test). ( f ) Nuclear extracts from cortical neurons transfected without (mock) or with control siRNA, L1 siRNA or TOP1 siRNA were incubated with supercoiled pUC19 plasmid DNA. For control, the vector was incubated with the reaction buffer. The samples were then subjected to agarose gel electrophoresis and ethidium bromide staining. A representative image of a stained gel is shown.

Techniques Used: Migration, Comparison, Control, Transfection, Incubation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

The L1/TOP1 interaction regulates the expression of the long autism genes Nrxn1 and Nlgn1 and the expression of the mitochondrial gene ND2. Cortical neurons were mock-transfected or transfected with L1 siRNA and subjected to RNA isolation and RT-qPCR 48 h after seeding. The mRNA levels of long autism ( a ) or mitochondrial ( b ) genes were normalized to the actin mRNA levels. ( c ) Cerebellar neurons from L1/858-863 mutant, L1/687 mutant and their wild-type littermates were maintained in culture for 48 h and subjected to RNA isolation and RT-qPCR. The ND2, Nrx1 and Nrx1 mRNA levels were normalized to the actin mRNA levels. Mean values + SD from five independent experiments with triplicates ( n = 5 mice) ( a , b ) or from six cultures per genotype ( n = 6 mice) ( c ) are shown for normalized mRNA levels in neurons after transfection with L1 siRNA relative to the levels in mock-transfected neurons ( a , b ) or in mutant neurons relative to wild-type neurons ( c ) (* p < 0.05, ** p < 0.01, *** p < 0.005; one-way ANOVA with Tukey’s ( a , b ) or Bonferroni’s ( c ) multiple comparison tests).

Figure Legend Snippet: The L1/TOP1 interaction regulates the expression of the long autism genes Nrxn1 and Nlgn1 and the expression of the mitochondrial gene ND2. Cortical neurons were mock-transfected or transfected with L1 siRNA and subjected to RNA isolation and RT-qPCR 48 h after seeding. The mRNA levels of long autism ( a ) or mitochondrial ( b ) genes were normalized to the actin mRNA levels. ( c ) Cerebellar neurons from L1/858-863 mutant, L1/687 mutant and their wild-type littermates were maintained in culture for 48 h and subjected to RNA isolation and RT-qPCR. The ND2, Nrx1 and Nrx1 mRNA levels were normalized to the actin mRNA levels. Mean values + SD from five independent experiments with triplicates ( n = 5 mice) ( a , b ) or from six cultures per genotype ( n = 6 mice) ( c ) are shown for normalized mRNA levels in neurons after transfection with L1 siRNA relative to the levels in mock-transfected neurons ( a , b ) or in mutant neurons relative to wild-type neurons ( c ) (* p < 0.05, ** p < 0.01, *** p < 0.005; one-way ANOVA with Tukey’s ( a , b ) or Bonferroni’s ( c ) multiple comparison tests).

Techniques Used: Expressing, Transfection, Isolation, Quantitative RT-PCR, Mutagenesis, Comparison

Control:

Article Title: The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions
Article Snippet: .. Mouse L1 siRNA (NCAM-L1; sc-43173), mouse TOP1 siRNA (Topo I; sc-36693), mouse PPARγ siRNA (PPARgamma; sc-29456), mouse NDUFV2 siRNA (sc-149892), and control siRNA (Control siRNA-A; sc-37007) were from Santa Cruz Biotechnology. .. FuGENE ® HD Transfection Reagent (Cat #E2311) was from Promega (Walldorf, Germany).

Image Search Results

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms24032097

Figure Lengend Snippet: L1-70 interacts with TOP1, PPARγ and NDUFV2, but not with other L1 binding partners in neurons. Cultured cortical neurons were treated with vehicle (-) or with the serine protease inhibitor aprotinin. They were then subjected to proximity ligation with L1 antibody and antibodies against SFPQ, NonO, PSPC1, WDR5, TOP1, HistH1, Nup93, Hsc70, SYT1, impβ1, ERα, RXR, PPARγ, NDUFV2, AR, VDR or GAPDH. ( a ) Representative images of vehicle- and aprotinin-treated neurons stained with the mouse L1 antibody C-2 and rabbit antibodies against TOP1, PPARγ or NDUFV2 are shown. Nuclei are stained with DAPI. Scale bars: 10 µm. ( b ) Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of vehicle control set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). ( c ) Cortical neurons were treated with or without L1 antibody 557. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatment without antibody 557 set to 100%). (*** p < 0.005; one-way ANOVA with Bonferroni’s multiple comparison test). ( d ) Cortical and cerebellar neurons pretreated with vehicle or aprotinin were incubated without or with antibody 557 in the absence or presence of aprotinin. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatments without aprotinin set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test).

Article Snippet: Mouse L1 siRNA (NCAM-L1; sc-43173), mouse TOP1 siRNA (Topo I; sc-36693), mouse PPARγ siRNA (PPARgamma; sc-29456), mouse NDUFV2 siRNA (sc-149892), and control siRNA (Control siRNA-A; sc-37007) were from Santa Cruz Biotechnology.

Techniques: Binding Assay, Cell Culture, Protease Inhibitor, Ligation, Staining, Control, Comparison, Incubation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms24032097

Figure Lengend Snippet: L1-ICD binds to TOP1 and PPARγ. Recombinant TOP1 ( a ) and PPARγ ( b ) were substrate-coated and incubated with increasing concentrations of L1-ICD. Binding of L1-ICD was determined by ELISA using mouse L1 antibody C-2 and horseradish peroxidase-conjugated secondary antibody. Mean values ± SD from three independent experiments carried out in triplicate are shown. ( c ) Recombinant TOP1 and PPARγ were substrate-coated and incubated with L1-ICD in the absence or presence of a five-fold excess of L1 peptides P1, P2, P3, P4 or P5, which cover the entire L1-ICD sequence. The ICD and transmembrane domain (TMD) of L1 and the positions of the peptides in L1-ICD are visualized. Binding was determined by ELISA using mouse L1 antibody C-2 and horseradish peroxidase-conjugated secondary antibody. Mean values + SD from five independent experiments carried out in triplicate are shown for the binding relative to control (values in the absence of peptides set to 100%) (** p < 0.01, *** p < 0.005, **** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test).

Techniques: Recombinant, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Control, Comparison

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms24032097

Figure Lengend Snippet: No enhanced interaction of L1 with TOP1, PPARγ and NDUFV2 in stimulated cortical neurons from L1-70-lacking mutant mice. Cultured cortical neurons from L1/687 ( a ) and L1/858–863 ( b ) mutant mice and their wild-type littermates ( a , b ) were treated without (-) or with L1 antibody 557 (557) and then subjected to proximity ligation assay with L1 antibody and TOP1, PPARγ or NDUFV2 antibodies. ( a ) For control, the L1 antibody and an antibody against SFPQ, ERα or RXR were used for proximity ligation. ( a , b ) Mean values + SD are shown for the average numbers of L1/TOP1-, L1/NDUFV2-, L1/PPARγ-, L1/SFPQ-, L1/ERα-, and L1/RXR-positive dots per cell relative to control (values of unstimulated wild-type neurons set to 100%) (** p < 0.01, **** p < 0.001 relative to non-stimulated wild-type neurons, ## p < 0.01, #### p < 0.001 relative to stimulated wild-type neurons; one-way ANOVA with Dunn’s multiple comparison test). ( c ) Non-nuclear brain fractions from wild-type (WT) and L1/858–863 mutant (Mut) mice were subjected to immunoprecipitation (IP) with immobilized mouse antibodies against TOP1, PPARγ and NDUFV2. Non-nuclear fractions (input) and immunoprecipitates were subjected to Western blot (WB) analysis with L1 antibody C-2. The arrowhead indicates L1-70.

Techniques: Mutagenesis, Cell Culture, Proximity Ligation Assay, Control, Ligation, Comparison, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms24032097

Figure Lengend Snippet: Reduction of TOP1, PPARγ or NDUFV2 expression inhibits L1-dependent neurite outgrowth. ( a , b ) Cortical neurons were mock-transfected or transfected with control siRNA, TOP1 siRNA, PPARγ siRNA or NDUFV2 siRNA. Neurons were then treated without or with L1 antibody 557. ( a , b ) Mean values + SEM from three independent experiments are shown for total neurite lengths (**** p < 0.001 relative to mock-transfected non-stimulated neurons, ### p < 0.005, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Dunn’s multiple comparison test).

Techniques: Expressing, Transfection, Control, Comparison

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms24032097

Figure Lengend Snippet: The interaction of L1 with TOP1, PPARγ or NDUFV2 is reduced by L1, TOP1, PPARγ and NDUFV2 siRNAs. ( a ) Cultured cortical neurons were mock-transfected or transfected with TOP1, PPARγ, NDUFV2 or L1 siRNAs. The transfected cells were then subjected to proximity ligation with L1 antibody and antibodies against TOP1, PPARγ or NDUFV2. Mean values + SD from two experiments are shown for the average numbers of L1/TOP1-, L1/PPARγ- and L1/NDUFV2-positive red dots per cell relative to control (values of mock-transfected neurons set to 100%) (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). ( b ) Western blot analysis of lysates from mock-transfected neurons or neurons transfected with TOP1 siRNA using TOP1 antibody. Ponceau S staining of a prominent 35 kDa band served as loading control. ( c ) Mock-transfected neurons or neurons transfected with PPARγ or NDUFV2 siRNAs were subjected to immunostaining with PPARγ or NDUFV2 antibody. Nuclei are stained with DAPI. Representative images are shown and indicate reduced PPARγ or NDUFV2 levels in neurons transfected with PPARγ siRNA or NDUFV2 siRNA. Scale bars: 10 µm.

Techniques: Cell Culture, Transfection, Ligation, Control, Comparison, Western Blot, Staining, Immunostaining

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms24032097

Figure Lengend Snippet: Disturbance of the L1/TOP1 and L1/PPARγ interactions inhibits L1-dependent neurite outgrowth from cortical neurons. ( a , b ) Cortical neurons were treated without (-) or with tat-P4 peptide. Neurons were then treated without or with L1 antibody 557 (557). ( a ) Mean values + SEM from three independent experiments are shown for total neurite lengths (**** p < 0.001 relative to mock-transfected non-stimulated neurons, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Dunn’s multiple comparison test). ( b ) After the treatments, neurons were subjected to proximity ligation with L1 antibody and TOP1 antibody or PPARγ antibody. Mean values + SD from three independent experiments are shown for average numbers of L1/TOP1- and L1/PPARγ-positive red dots per cell relative to control (values of non-transfected neurons set to 100%) (*** p < 0.005, **** p < 0.001 relative to mock-transfected stimulated neurons, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Bonferroni’s multiple comparison test).

Techniques: Transfection, Comparison, Ligation, Control

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms24032097

Figure Lengend Snippet: Topoisomerase inhibitors reduce L1-dependent neurite outgrowth as well as neuronal survival and migration. Cerebellar neurons ( a , c ) and cerebellar explants ( e ), and cortical neurons ( b , d ) were treated without (-) or with topotecan (Topo) or irinotecan (Irino) in the absence or presence of L1 antibody 557. ( a , b ) Mean values + SEM from three independent experiments are shown for total neurite lengths (*** p < 0.005, **** p < 0.001 relative to non-stimulated neurons in the absence of inhibitors, ### p < 0.005, #### p < 0.001 relative to stimulated neurons in the absence of inhibitors; one-way ANOVA with Dunn’s multiple comparison test). ( c , d ) Mean values + SEM from three independent experiments are shown for the numbers of viable cells relative to control (values of unstimulated neurons in the absence of H 2 O 2 set to 100%) (#### p < 0.0001 relative to stimulated neurons in absence of inhibitors and the presence of H 2 O 2 , **** p < 0.0001 relative to non-stimulated neurons in absence of inhibitors and the presence of H 2 O 2 , §§§§ p < 0.0001 relative to non-stimulated neurons in absence of inhibitors and H 2 O 2 ; one-way ANOVA with Dunn’s multiple comparison test). ( e ) Mean values + SEM from three independent experiments are shown for the total numbers of migrating cells (### p < 0.005 relative to stimulated neurons in absence of inhibitors, *** p < 0.005 relative to non-stimulated neurons in absence of inhibitors; one-way ANOVA with Dunn’s multiple comparison test). ( f ) Nuclear extracts from cortical neurons transfected without (mock) or with control siRNA, L1 siRNA or TOP1 siRNA were incubated with supercoiled pUC19 plasmid DNA. For control, the vector was incubated with the reaction buffer. The samples were then subjected to agarose gel electrophoresis and ethidium bromide staining. A representative image of a stained gel is shown.

Techniques: Migration, Comparison, Control, Transfection, Incubation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms24032097

Figure Lengend Snippet: The L1/TOP1 interaction regulates the expression of the long autism genes Nrxn1 and Nlgn1 and the expression of the mitochondrial gene ND2. Cortical neurons were mock-transfected or transfected with L1 siRNA and subjected to RNA isolation and RT-qPCR 48 h after seeding. The mRNA levels of long autism ( a ) or mitochondrial ( b ) genes were normalized to the actin mRNA levels. ( c ) Cerebellar neurons from L1/858-863 mutant, L1/687 mutant and their wild-type littermates were maintained in culture for 48 h and subjected to RNA isolation and RT-qPCR. The ND2, Nrx1 and Nrx1 mRNA levels were normalized to the actin mRNA levels. Mean values + SD from five independent experiments with triplicates ( n = 5 mice) ( a , b ) or from six cultures per genotype ( n = 6 mice) ( c ) are shown for normalized mRNA levels in neurons after transfection with L1 siRNA relative to the levels in mock-transfected neurons ( a , b ) or in mutant neurons relative to wild-type neurons ( c ) (* p < 0.05, ** p < 0.01, *** p < 0.005; one-way ANOVA with Tukey’s ( a , b ) or Bonferroni’s ( c ) multiple comparison tests).

Techniques: Expressing, Transfection, Isolation, Quantitative RT-PCR, Mutagenesis, Comparison

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mouse top1 sirna (Santa Cruz Biotechnology)

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1) Product Images from "The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions"

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