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empty plpc vector  (Addgene inc)


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    Addgene inc empty plpc vector
    REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 <t>cells</t> <t>(transfected</t> with empty vector <t>pLPC</t> vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant
    Empty Plpc Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/empty+plpc+vector/pLPC-N+FLAG+(Plasmid+%2312521)/pmc11015551-415-17-20
    Average 93 stars, based on 11 article reviews
    empty plpc vector - by Bioz Stars, 2026-11
    93/100 stars

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    1) Product Images from "Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells"

    Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

    Journal: BMC Biology

    doi: 10.1186/s12915-024-01879-0

    REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant
    Figure Legend Snippet: REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant

    Techniques Used: Expressing, Western Blot, Clone Assay, Comparison, Transfection, Plasmid Preparation, Two Tailed Test, Wound Healing Assay, Marker, CRISPR, Derivative Assay, One-tailed Test

    Related Articles

    Transfection:

    Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells
    Article Snippet: .. On the next day, cells were transiently transfected with 500 ng either REST-WT-expressing pLPC-vector (Addgene, #41903) or empty pLPC vector (Addgene, #12521) with Fugene HD transfection reagent (Promega) following manufacturer’s instructions. ..

    Clone Assay:

    Article Title: Phosphatase activity is dispensable for PRL-3-mediated oncogenesis and tumor progression
    Article Snippet: Site-directed mutagenesis (Agilent QuikChange Lightning, 210518) was used to generate PRL-3 mutants (see for primers). .. Entry clones were recombined into Rag2.GW.DEST , pLPC-N FLAG (RRID:Addgene_12521), pLenti-CMV-Puro-DEST (RRID:Addgene_17452), or pCW57.1 (RRID:Addgene_41393) using LR Clonase II (Thermo Fisher, 11791020). ..

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: The 3×HA and 3×HA-GFP sequences were cloned from pMXs-3XHA-EGFP-OMP25 plasmid (Addgene 83356). pLJC5-3XHA-EGFP-PEX26 was a gift from David Sabatini (Addgene, 139054; http://n2t.net/addgene:139054 ). .. The DNA sequence of 3XHA-EGFP-PEX26 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) restriction digestion, gene-specific PCR and Quick Ligation Kit (NEB, M2200S). .. For the proteoliposome, DNA sequences of SLC45A4 or EGFP-OMP25 (derived from Addgene, 83356) were cloned to pEU-E01 vector (CellFree Sciences) using EcoRV-HF (NEB, R3136S) and Q5® Hot Start High-Fidelity DNA Polymerase (NEB, M0493) with appropriate primers.

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis.
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: SLC45A4 encodes a mitochondrial putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1 ) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis.
    Article Snippet: The 3×HA and 3×HA-GFP sequences were cloned from pMXs-3XHA-EGFP-OMP25 plasmid (Addgene 83356). pLJC5-3XHA-EGFP-PEX26was a gift fromDavid Sabatini (Addgene, 139054; http://n2t.net/addgene:139054). .. The DNA sequence of 3XHAEGFP-PEX26was cloned to pLPC-NFLAGvector (Addgene, 12521) using BamHI-HF (NEB, R3136S) restriction digestion, gene-specific PCR and Quick Ligation Kit (NEB, M2200S). .. For the proteoliposome, DNA sequences of SLC45A4 or EGFPOMP25 (derived fromAddgene, 83356) were cloned to pEU-E01 vector (CellFree Sciences) using EcoRV-HF (NEB, R3136S) and Q5® Hot Start High-Fidelity DNA Polymerase (NEB, M0493) with appropriate primers.

    Sequencing:

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: The 3×HA and 3×HA-GFP sequences were cloned from pMXs-3XHA-EGFP-OMP25 plasmid (Addgene 83356). pLJC5-3XHA-EGFP-PEX26 was a gift from David Sabatini (Addgene, 139054; http://n2t.net/addgene:139054 ). .. The DNA sequence of 3XHA-EGFP-PEX26 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) restriction digestion, gene-specific PCR and Quick Ligation Kit (NEB, M2200S). .. For the proteoliposome, DNA sequences of SLC45A4 or EGFP-OMP25 (derived from Addgene, 83356) were cloned to pEU-E01 vector (CellFree Sciences) using EcoRV-HF (NEB, R3136S) and Q5® Hot Start High-Fidelity DNA Polymerase (NEB, M0493) with appropriate primers.

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis.
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: The co-receptor Tspan12 directly captures Norrin to promote ligand-specific β-catenin signaling
    Article Snippet: .. For expression in Sf9 cells, full-length human Tspan12 (DNASU), Tspan12ΔC (residues 1-252), and Tspan12 with the large extracellular loop replaced by that of TSPAN11 (Addgene Plasmid #115785 from Harald Junge ( )) were C-terminally tagged with the Rho-1D4 antibody recognition sequence TETSQVAPA. ..

    Article Title: SLC45A4 encodes a mitochondrial putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1 ) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis.
    Article Snippet: The 3×HA and 3×HA-GFP sequences were cloned from pMXs-3XHA-EGFP-OMP25 plasmid (Addgene 83356). pLJC5-3XHA-EGFP-PEX26was a gift fromDavid Sabatini (Addgene, 139054; http://n2t.net/addgene:139054). .. The DNA sequence of 3XHAEGFP-PEX26was cloned to pLPC-NFLAGvector (Addgene, 12521) using BamHI-HF (NEB, R3136S) restriction digestion, gene-specific PCR and Quick Ligation Kit (NEB, M2200S). .. For the proteoliposome, DNA sequences of SLC45A4 or EGFPOMP25 (derived fromAddgene, 83356) were cloned to pEU-E01 vector (CellFree Sciences) using EcoRV-HF (NEB, R3136S) and Q5® Hot Start High-Fidelity DNA Polymerase (NEB, M0493) with appropriate primers.

    Plasmid Preparation:

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: The 3×HA and 3×HA-GFP sequences were cloned from pMXs-3XHA-EGFP-OMP25 plasmid (Addgene 83356). pLJC5-3XHA-EGFP-PEX26 was a gift from David Sabatini (Addgene, 139054; http://n2t.net/addgene:139054 ). .. The DNA sequence of 3XHA-EGFP-PEX26 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) restriction digestion, gene-specific PCR and Quick Ligation Kit (NEB, M2200S). .. For the proteoliposome, DNA sequences of SLC45A4 or EGFP-OMP25 (derived from Addgene, 83356) were cloned to pEU-E01 vector (CellFree Sciences) using EcoRV-HF (NEB, R3136S) and Q5® Hot Start High-Fidelity DNA Polymerase (NEB, M0493) with appropriate primers.

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis.
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: The co-receptor Tspan12 directly captures Norrin to promote ligand-specific β-catenin signaling
    Article Snippet: .. For expression in Sf9 cells, full-length human Tspan12 (DNASU), Tspan12ΔC (residues 1-252), and Tspan12 with the large extracellular loop replaced by that of TSPAN11 (Addgene Plasmid #115785 from Harald Junge ( )) were C-terminally tagged with the Rho-1D4 antibody recognition sequence TETSQVAPA. ..

    Article Title: SLC45A4 encodes a mitochondrial putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1 ) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Polymerase Chain Reaction:

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: The 3×HA and 3×HA-GFP sequences were cloned from pMXs-3XHA-EGFP-OMP25 plasmid (Addgene 83356). pLJC5-3XHA-EGFP-PEX26 was a gift from David Sabatini (Addgene, 139054; http://n2t.net/addgene:139054 ). .. The DNA sequence of 3XHA-EGFP-PEX26 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) restriction digestion, gene-specific PCR and Quick Ligation Kit (NEB, M2200S). .. For the proteoliposome, DNA sequences of SLC45A4 or EGFP-OMP25 (derived from Addgene, 83356) were cloned to pEU-E01 vector (CellFree Sciences) using EcoRV-HF (NEB, R3136S) and Q5® Hot Start High-Fidelity DNA Polymerase (NEB, M0493) with appropriate primers.

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis.
    Article Snippet: The 3×HA and 3×HA-GFP sequences were cloned from pMXs-3XHA-EGFP-OMP25 plasmid (Addgene 83356). pLJC5-3XHA-EGFP-PEX26was a gift fromDavid Sabatini (Addgene, 139054; http://n2t.net/addgene:139054). .. The DNA sequence of 3XHAEGFP-PEX26was cloned to pLPC-NFLAGvector (Addgene, 12521) using BamHI-HF (NEB, R3136S) restriction digestion, gene-specific PCR and Quick Ligation Kit (NEB, M2200S). .. For the proteoliposome, DNA sequences of SLC45A4 or EGFPOMP25 (derived fromAddgene, 83356) were cloned to pEU-E01 vector (CellFree Sciences) using EcoRV-HF (NEB, R3136S) and Q5® Hot Start High-Fidelity DNA Polymerase (NEB, M0493) with appropriate primers.

    Ligation:

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: The 3×HA and 3×HA-GFP sequences were cloned from pMXs-3XHA-EGFP-OMP25 plasmid (Addgene 83356). pLJC5-3XHA-EGFP-PEX26 was a gift from David Sabatini (Addgene, 139054; http://n2t.net/addgene:139054 ). .. The DNA sequence of 3XHA-EGFP-PEX26 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) restriction digestion, gene-specific PCR and Quick Ligation Kit (NEB, M2200S). .. For the proteoliposome, DNA sequences of SLC45A4 or EGFP-OMP25 (derived from Addgene, 83356) were cloned to pEU-E01 vector (CellFree Sciences) using EcoRV-HF (NEB, R3136S) and Q5® Hot Start High-Fidelity DNA Polymerase (NEB, M0493) with appropriate primers.

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis.
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: SLC45A4 encodes a mitochondrial putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis
    Article Snippet: Human cDNA encoding SLC45A4 (NCBI: NM_001286646.1 ) was purchased from GenScript. .. The DNA sequence of SLC45A4 was cloned to pLPC-N FLAG vector (Addgene, 12521) using BamHI-HF (NEB, R3136S) and EcoRI-HF (NEB, R3101S) restriction digestion and Quick Ligation Kit (NEB, M2200S). .. Sequences coding 3×HA or 3×HA-GFP tags were added to the C-terminal of the protein using gene-specific PCR and HiFi assembly (NEB).

    Article Title: SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis.
    Article Snippet: The 3×HA and 3×HA-GFP sequences were cloned from pMXs-3XHA-EGFP-OMP25 plasmid (Addgene 83356). pLJC5-3XHA-EGFP-PEX26was a gift fromDavid Sabatini (Addgene, 139054; http://n2t.net/addgene:139054). .. The DNA sequence of 3XHAEGFP-PEX26was cloned to pLPC-NFLAGvector (Addgene, 12521) using BamHI-HF (NEB, R3136S) restriction digestion, gene-specific PCR and Quick Ligation Kit (NEB, M2200S). .. For the proteoliposome, DNA sequences of SLC45A4 or EGFPOMP25 (derived fromAddgene, 83356) were cloned to pEU-E01 vector (CellFree Sciences) using EcoRV-HF (NEB, R3136S) and Q5® Hot Start High-Fidelity DNA Polymerase (NEB, M0493) with appropriate primers.

    Expressing:

    Article Title: The co-receptor Tspan12 directly captures Norrin to promote ligand-specific β-catenin signaling
    Article Snippet: .. For expression in Sf9 cells, full-length human Tspan12 (DNASU), Tspan12ΔC (residues 1-252), and Tspan12 with the large extracellular loop replaced by that of TSPAN11 (Addgene Plasmid #115785 from Harald Junge ( )) were C-terminally tagged with the Rho-1D4 antibody recognition sequence TETSQVAPA. ..



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    91
    Addgene inc empty vector control plpc
    REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 <t>cells</t> <t>(transfected</t> with empty vector <t>pLPC</t> vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant
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    REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant

    Journal: BMC Biology

    Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

    doi: 10.1186/s12915-024-01879-0

    Figure Lengend Snippet: REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant

    Article Snippet: On the next day, cells were transiently transfected with 500 ng either REST-WT-expressing pLPC-vector (Addgene, #41903) or empty pLPC vector (Addgene, #12521) with Fugene HD transfection reagent (Promega) following manufacturer’s instructions.

    Techniques: Expressing, Western Blot, Clone Assay, Comparison, Transfection, Plasmid Preparation, Two Tailed Test, Wound Healing Assay, Marker, CRISPR, Derivative Assay, One-tailed Test