hek293t cells  (TaKaRa)


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    TaKaRa hek293t cells
    Hek293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hek293t cells  (TaKaRa)


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    TaKaRa hek293t cells
    (A) Condensate formation of DRACC1 mutants. <t>HEK293T</t> cells were transfected with indicated DRACC1 mutants. 24 hrs later, the cells were fixed and observed with a confocal microscope. (B) Primary structure of mouse DRACC1. The coiled-coil domains (CC1 and CC2) and low complexity region (LCR) were identified using SMART. Evolutional conservation was analyzed using ConSurf. The probability of disorder is assessed using IUPred2A. Truncate mutants described at the bottom were used in (A). (C-E) Quantitative analysis of DRACC1 droplets described in (A). Number of DRACC1 condensates per cell (C), and size of DRACC1 condensates (D) Maximum size of DRACC1 condensates in individual cells (E) are described. The numbers of analyzed cells expressing full length, Δ1-36, Δ37-74, Δ75-106, Δ107-157, Δ158-187, N-half, or C-half DRACC1 is 98, 85, 87, 109, 95, 101, 93, 83, respectively. (F) Impaired PSD localization of the condensation deficient DRACC1 mutant. Primary cultured mouse hippocampal neurons were transfected with full-length or Δ107-157 DRACC1 mutants together with DsRed at DIV19. 2 days later, the cells were fixed and observed with a confocal microscope. Scale bars: 50 μm (white) or 10 μm (yellow). *P < 0.05
    Hek293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "DRACC1, a major postsynaptic protein, regulates the condensation of postsynaptic proteins via liquid-liquid phase separation"

    Article Title: DRACC1, a major postsynaptic protein, regulates the condensation of postsynaptic proteins via liquid-liquid phase separation

    Journal: bioRxiv

    doi: 10.1101/2023.01.23.525126

    (A) Condensate formation of DRACC1 mutants. HEK293T cells were transfected with indicated DRACC1 mutants. 24 hrs later, the cells were fixed and observed with a confocal microscope. (B) Primary structure of mouse DRACC1. The coiled-coil domains (CC1 and CC2) and low complexity region (LCR) were identified using SMART. Evolutional conservation was analyzed using ConSurf. The probability of disorder is assessed using IUPred2A. Truncate mutants described at the bottom were used in (A). (C-E) Quantitative analysis of DRACC1 droplets described in (A). Number of DRACC1 condensates per cell (C), and size of DRACC1 condensates (D) Maximum size of DRACC1 condensates in individual cells (E) are described. The numbers of analyzed cells expressing full length, Δ1-36, Δ37-74, Δ75-106, Δ107-157, Δ158-187, N-half, or C-half DRACC1 is 98, 85, 87, 109, 95, 101, 93, 83, respectively. (F) Impaired PSD localization of the condensation deficient DRACC1 mutant. Primary cultured mouse hippocampal neurons were transfected with full-length or Δ107-157 DRACC1 mutants together with DsRed at DIV19. 2 days later, the cells were fixed and observed with a confocal microscope. Scale bars: 50 μm (white) or 10 μm (yellow). *P < 0.05
    Figure Legend Snippet: (A) Condensate formation of DRACC1 mutants. HEK293T cells were transfected with indicated DRACC1 mutants. 24 hrs later, the cells were fixed and observed with a confocal microscope. (B) Primary structure of mouse DRACC1. The coiled-coil domains (CC1 and CC2) and low complexity region (LCR) were identified using SMART. Evolutional conservation was analyzed using ConSurf. The probability of disorder is assessed using IUPred2A. Truncate mutants described at the bottom were used in (A). (C-E) Quantitative analysis of DRACC1 droplets described in (A). Number of DRACC1 condensates per cell (C), and size of DRACC1 condensates (D) Maximum size of DRACC1 condensates in individual cells (E) are described. The numbers of analyzed cells expressing full length, Δ1-36, Δ37-74, Δ75-106, Δ107-157, Δ158-187, N-half, or C-half DRACC1 is 98, 85, 87, 109, 95, 101, 93, 83, respectively. (F) Impaired PSD localization of the condensation deficient DRACC1 mutant. Primary cultured mouse hippocampal neurons were transfected with full-length or Δ107-157 DRACC1 mutants together with DsRed at DIV19. 2 days later, the cells were fixed and observed with a confocal microscope. Scale bars: 50 μm (white) or 10 μm (yellow). *P < 0.05

    Techniques Used: Transfection, Microscopy, Expressing, Mutagenesis, Cell Culture

    (A-D) Live-imaging of HEK293T cells transfected with DRACC1-GFP. (A) Overview of the observed cells. Regions magnified in panels B and C are described. See also Supplementary Movie 2. (B and C) Representative movie images of punctate structures undergoing fusion (B) and fission (C). (D) Enlarged rigid structure with a stable shape. See also Supplementary Movie 3. (E) The effect of 1,6-hexanediol on DRACC1 positive structures. HEK293T cells were transfected with DRACC1-GFP. 24 hrs later, the medium was changed to fresh medium, including the indicated concentration of 1,6-hexanediol. 10 min later, the cells were fixed. (F and G) Interaction between DRACC1 or interaction of DRACC1 and PSD-95, SynGAP, or GluN2B. HEK293T cells were transfected with indicated plasmids. 24 hrs later, cells were lysed, and immunoprecipitation was performed with an anti-FLAG antibody. Then, immunoblotting was performed with anti-GFP or anti-FLAG antibodies. (H-J) Localization of DRACC1 on SynGAP-positive droplets. HEK293T cells were transfected with indicated plasmids. 24 hrs later, cells were fixed and observed with confocal microscopy. For panels H and J, cells were subjected to immunocytochemistry using an anti-FLAG antibody before observation. Scale bars: 10 μm (white) or 5 μm (yellow) in (A-D), 50 μm (white) or 10 μm (yellow) in (E), and 20 μm (white) or 4 μm (yellow) in (H-J), respectively.
    Figure Legend Snippet: (A-D) Live-imaging of HEK293T cells transfected with DRACC1-GFP. (A) Overview of the observed cells. Regions magnified in panels B and C are described. See also Supplementary Movie 2. (B and C) Representative movie images of punctate structures undergoing fusion (B) and fission (C). (D) Enlarged rigid structure with a stable shape. See also Supplementary Movie 3. (E) The effect of 1,6-hexanediol on DRACC1 positive structures. HEK293T cells were transfected with DRACC1-GFP. 24 hrs later, the medium was changed to fresh medium, including the indicated concentration of 1,6-hexanediol. 10 min later, the cells were fixed. (F and G) Interaction between DRACC1 or interaction of DRACC1 and PSD-95, SynGAP, or GluN2B. HEK293T cells were transfected with indicated plasmids. 24 hrs later, cells were lysed, and immunoprecipitation was performed with an anti-FLAG antibody. Then, immunoblotting was performed with anti-GFP or anti-FLAG antibodies. (H-J) Localization of DRACC1 on SynGAP-positive droplets. HEK293T cells were transfected with indicated plasmids. 24 hrs later, cells were fixed and observed with confocal microscopy. For panels H and J, cells were subjected to immunocytochemistry using an anti-FLAG antibody before observation. Scale bars: 10 μm (white) or 5 μm (yellow) in (A-D), 50 μm (white) or 10 μm (yellow) in (E), and 20 μm (white) or 4 μm (yellow) in (H-J), respectively.

    Techniques Used: Imaging, Transfection, Concentration Assay, Immunoprecipitation, Western Blot, Confocal Microscopy, Immunocytochemistry

    human hek 293t cells  (TaKaRa)


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    TaKaRa human hek 293t cells
    Human Hek 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hek293t lenti x cells  (TaKaRa)


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    TaKaRa hek293t lenti x cells
    ( A – C ) Barcode-specific gRNA-dependent activation of EGFP reporters for two barcoded <t>HEK293T</t> strains established for each of CloneSelect C→T ( A ), low-copy CRISPRa ( B ), and high-copy CRISPRa ( C ) (n=3). Scale bar, 50 μm. Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. ( D ) Median EGFP intensities of genome editing-activated EGFP positive cells (n=3). The Mann-Whitney U test was performed to compare two groups. ( E ) Comparison of Target-AID variants and a nCas9 (D10A) control in the CloneSelect C→T reporter activation for the same set of barcode-gRNA pairs (n=1). Welch’s t-test was performed to compare OT and NT activations. ( F ) Reporter activation in HeLa cells by CloneSelect C→T (n=3). Welch’s t-test was performed to compare OT and NT activations. Scale bar, 80 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.
    Hek293t Lenti X Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A multi-kingdom genetic barcoding system for precise target clone isolation"

    Article Title: A multi-kingdom genetic barcoding system for precise target clone isolation

    Journal: bioRxiv

    doi: 10.1101/2023.01.18.524633

    ( A – C ) Barcode-specific gRNA-dependent activation of EGFP reporters for two barcoded HEK293T strains established for each of CloneSelect C→T ( A ), low-copy CRISPRa ( B ), and high-copy CRISPRa ( C ) (n=3). Scale bar, 50 μm. Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. ( D ) Median EGFP intensities of genome editing-activated EGFP positive cells (n=3). The Mann-Whitney U test was performed to compare two groups. ( E ) Comparison of Target-AID variants and a nCas9 (D10A) control in the CloneSelect C→T reporter activation for the same set of barcode-gRNA pairs (n=1). Welch’s t-test was performed to compare OT and NT activations. ( F ) Reporter activation in HeLa cells by CloneSelect C→T (n=3). Welch’s t-test was performed to compare OT and NT activations. Scale bar, 80 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.
    Figure Legend Snippet: ( A – C ) Barcode-specific gRNA-dependent activation of EGFP reporters for two barcoded HEK293T strains established for each of CloneSelect C→T ( A ), low-copy CRISPRa ( B ), and high-copy CRISPRa ( C ) (n=3). Scale bar, 50 μm. Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. ( D ) Median EGFP intensities of genome editing-activated EGFP positive cells (n=3). The Mann-Whitney U test was performed to compare two groups. ( E ) Comparison of Target-AID variants and a nCas9 (D10A) control in the CloneSelect C→T reporter activation for the same set of barcode-gRNA pairs (n=1). Welch’s t-test was performed to compare OT and NT activations. ( F ) Reporter activation in HeLa cells by CloneSelect C→T (n=3). Welch’s t-test was performed to compare OT and NT activations. Scale bar, 80 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.

    Techniques Used: Activation Assay, MANN-WHITNEY

    ( A ) Barcode-specific gRNA-dependent reporter activation circuit using wild-type Cas9. ( B ) Barcode-specific activation of the deletion-based reporter prepared for two barcodes (BC-del1 and BC-del2) in HEK293T cells (n=3). Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 50 μm. ( C ) ROC curves along varying reporter intensity thresholds for target barcoded cells (n=3).
    Figure Legend Snippet: ( A ) Barcode-specific gRNA-dependent reporter activation circuit using wild-type Cas9. ( B ) Barcode-specific activation of the deletion-based reporter prepared for two barcodes (BC-del1 and BC-del2) in HEK293T cells (n=3). Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 50 μm. ( C ) ROC curves along varying reporter intensity thresholds for target barcoded cells (n=3).

    Techniques Used: Activation Assay

    ( A ) scCloneSelect. ( B and C ) Barcode-specific gRNA-dependent reporter activation of the original CloneSelect C→T and scCloneSelect in HEK293T cells (n=3). Scale bar, 50 μm. ( D and E ) Barcode-specific gRNA-dependent reporter activation of three barcoded mESC lines by scCloneSelect. Target-AID was stably integrated prior to the barcoding. gRNAs were delivered by lentiviral transduction. Scale bar, 100 μm. Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. * P < 0.05; ** P < 0.01; *** P < 0.001. ( F ) Schematic diagram of a scCloneSelect workflow to retrospectively isolate a cell clone demonstrating a gene expression profile of interest from a cell population stored before they demonstrate the target gene expression pattern. ( G ) mESC cell culture assays and clone isolation performed in this work. ( H ) scRNA-seq of mESC populations treated with LIF and 2i and those without LIF or 2i. ( I ) Distribution of cells for arbitrarily selected clones in the two-dimensional embedding of high-dimensional gene expression space by UMAP (uniform manifold approximation and projection). ( J ) Abundance of barcoded cell clones in the mESC population. The data was generated based on dntags identified by reamplifying the dntag reads from the original scRNA-seq libraries. ( K ) gRNA-specific activation of target barcoded clones in the mESC population. Scale bar, 50 μm. ( L ) Barcode enrichment analysis after cell sorting of the reporter-activated cells. Each row represents the barcode enrichment profile for each target isolation assay. The left heatmap was expanded from the dashed box area of the right heatmap.
    Figure Legend Snippet: ( A ) scCloneSelect. ( B and C ) Barcode-specific gRNA-dependent reporter activation of the original CloneSelect C→T and scCloneSelect in HEK293T cells (n=3). Scale bar, 50 μm. ( D and E ) Barcode-specific gRNA-dependent reporter activation of three barcoded mESC lines by scCloneSelect. Target-AID was stably integrated prior to the barcoding. gRNAs were delivered by lentiviral transduction. Scale bar, 100 μm. Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. * P < 0.05; ** P < 0.01; *** P < 0.001. ( F ) Schematic diagram of a scCloneSelect workflow to retrospectively isolate a cell clone demonstrating a gene expression profile of interest from a cell population stored before they demonstrate the target gene expression pattern. ( G ) mESC cell culture assays and clone isolation performed in this work. ( H ) scRNA-seq of mESC populations treated with LIF and 2i and those without LIF or 2i. ( I ) Distribution of cells for arbitrarily selected clones in the two-dimensional embedding of high-dimensional gene expression space by UMAP (uniform manifold approximation and projection). ( J ) Abundance of barcoded cell clones in the mESC population. The data was generated based on dntags identified by reamplifying the dntag reads from the original scRNA-seq libraries. ( K ) gRNA-specific activation of target barcoded clones in the mESC population. Scale bar, 50 μm. ( L ) Barcode enrichment analysis after cell sorting of the reporter-activated cells. Each row represents the barcode enrichment profile for each target isolation assay. The left heatmap was expanded from the dashed box area of the right heatmap.

    Techniques Used: Activation Assay, Stable Transfection, Transduction, Expressing, Cell Culture, Isolation, Clone Assay, Generated, FACS

    ( A ) EGFP-positive control expressions for the original CloneSelect C→T and scCloneSelect in HEK293T cells with the same genome editing conditions tested for the respective reporters (n=3). Scale bar, 50 μm. ( B ) Median EGFP intensities of base editing-activated EGFP positive cells (n=3). ( C and D ) Barcode-specific gRNA-dependent reporter activation of six barcoded cell lines by scCloneSelect (n=1). Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 50 μm. ( E ) RT-PCR of the scCloneSelect dntags in HEK293T. ( F ) Fraction of mESC single-cell transcriptome profiles (Drop-seq) that contained dntags and fraction of dntags reported in the uptag-dntag combination reference database. ( G ) Schematic representation of a scCloneSelect reporter activation assay where Target-AID was stably introduced to the cell population prior to barcoding and gRNA-dependent reporter activation. ( H and I ) gRNA-dependent reporter activation of target barcoded mESCs and CA1 hPSCs by scCloneSelect (n=2). Target-AID was stably integrated prior to the barcoding. Targeting gRNAs were delivered by transfection. Welch’s t-test was performed to compare OT and NT activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 100 μm. ( J ) Schematic representation of a scCloneSelect reporter activation assay where the target gRNA and Target-AID were electroporated together to the barcoded cell population. ( K ) gRNA-dependent reporter activation of barcoded H1 hPSCs by scCloneSelect (n=2). Targeting gRNA and Target-AID were electroporated together. Scale bar, 100 μm.
    Figure Legend Snippet: ( A ) EGFP-positive control expressions for the original CloneSelect C→T and scCloneSelect in HEK293T cells with the same genome editing conditions tested for the respective reporters (n=3). Scale bar, 50 μm. ( B ) Median EGFP intensities of base editing-activated EGFP positive cells (n=3). ( C and D ) Barcode-specific gRNA-dependent reporter activation of six barcoded cell lines by scCloneSelect (n=1). Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 50 μm. ( E ) RT-PCR of the scCloneSelect dntags in HEK293T. ( F ) Fraction of mESC single-cell transcriptome profiles (Drop-seq) that contained dntags and fraction of dntags reported in the uptag-dntag combination reference database. ( G ) Schematic representation of a scCloneSelect reporter activation assay where Target-AID was stably introduced to the cell population prior to barcoding and gRNA-dependent reporter activation. ( H and I ) gRNA-dependent reporter activation of target barcoded mESCs and CA1 hPSCs by scCloneSelect (n=2). Target-AID was stably integrated prior to the barcoding. Targeting gRNAs were delivered by transfection. Welch’s t-test was performed to compare OT and NT activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 100 μm. ( J ) Schematic representation of a scCloneSelect reporter activation assay where the target gRNA and Target-AID were electroporated together to the barcoded cell population. ( K ) gRNA-dependent reporter activation of barcoded H1 hPSCs by scCloneSelect (n=2). Targeting gRNA and Target-AID were electroporated together. Scale bar, 100 μm.

    Techniques Used: Positive Control, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection

    hek293t cells  (TaKaRa)


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    TaKaRa hek293t cells
    Hek293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    hek293t cells  (TaKaRa)


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    TaKaRa hek293t cells
    Fractionation of core particles by CsCl density gradient ultracentrifugation in transiently transfected <t>HEK293T</t> cells ( a ), and stably expressing HepG2/HisCore ( b ). The fractionation number (1–14) corresponds to the number of the blot. HBe antigen peaks were determined by ELISA. In all cells, core particles were detected in fractions with a density of around 1.26. An anti-core antibody (bottom panel) detected the core protein in the fraction. ( c ) Purification of HisCore or HisLacZ with Ni-NTA agarose. HisCore and HisLacZ (as a control) were detected in the bound fraction. For the detection of the HisCore protein, an anti-Core antibody was used. For the detection of the HisLacZ protein, an anti-His-tag antibody was used. Input (In), Unbound (Un), Bound (B).
    Hek293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification of the Interaction between Minichromosome Maintenance Proteins and the Core Protein of Hepatitis B Virus"

    Article Title: Identification of the Interaction between Minichromosome Maintenance Proteins and the Core Protein of Hepatitis B Virus

    Journal: Current Issues in Molecular Biology

    doi: 10.3390/cimb45010050

    Fractionation of core particles by CsCl density gradient ultracentrifugation in transiently transfected HEK293T cells ( a ), and stably expressing HepG2/HisCore ( b ). The fractionation number (1–14) corresponds to the number of the blot. HBe antigen peaks were determined by ELISA. In all cells, core particles were detected in fractions with a density of around 1.26. An anti-core antibody (bottom panel) detected the core protein in the fraction. ( c ) Purification of HisCore or HisLacZ with Ni-NTA agarose. HisCore and HisLacZ (as a control) were detected in the bound fraction. For the detection of the HisCore protein, an anti-Core antibody was used. For the detection of the HisLacZ protein, an anti-His-tag antibody was used. Input (In), Unbound (Un), Bound (B).
    Figure Legend Snippet: Fractionation of core particles by CsCl density gradient ultracentrifugation in transiently transfected HEK293T cells ( a ), and stably expressing HepG2/HisCore ( b ). The fractionation number (1–14) corresponds to the number of the blot. HBe antigen peaks were determined by ELISA. In all cells, core particles were detected in fractions with a density of around 1.26. An anti-core antibody (bottom panel) detected the core protein in the fraction. ( c ) Purification of HisCore or HisLacZ with Ni-NTA agarose. HisCore and HisLacZ (as a control) were detected in the bound fraction. For the detection of the HisCore protein, an anti-Core antibody was used. For the detection of the HisLacZ protein, an anti-His-tag antibody was used. Input (In), Unbound (Un), Bound (B).

    Techniques Used: Fractionation, Transfection, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Purification

    Pull-down assay of HisCore with recombinant Halo-MCM2. Halo-tagged MCM2 specifically interacted with the HisCore together with the associated MCMs. Cell lysates of HepG2/HisCore (lane 1), and HepG2/HisLacZ (lane 2) were prepared. The lysate was also prepared from HaloMCM2 transiently expressing HEK293T cells (lane 3). The cell lysate of HEK293T/HaloMCM2 was mixed with the lysate of either HepG2/HisCore or HepG2/HisLacZ and incubated at 4 °C overnight. The bound fraction was washed and eluted with an elution buffer and analyzed by Western blotting. Lane 1, input of HepG2/HisCore; lane 2, input of HepG2/HisLacZ; lane 3, input of HEK293T/HaloMCM2; lane 4, HaloMCM2-bound fraction with HepG2/HisCore; and lane 5, HaloMCM2-bound fraction with HepG2/HisLacZ. “+” indicates the use of those listed to the right. HisCore and HisLacZ proteins were detected by an anti-His-tag antibody. HaloMCM2 was detected by an anti-Halo™-tag antibody. Endogenous MCMs were detected by specific antibodies, as described in Materials and Methods.
    Figure Legend Snippet: Pull-down assay of HisCore with recombinant Halo-MCM2. Halo-tagged MCM2 specifically interacted with the HisCore together with the associated MCMs. Cell lysates of HepG2/HisCore (lane 1), and HepG2/HisLacZ (lane 2) were prepared. The lysate was also prepared from HaloMCM2 transiently expressing HEK293T cells (lane 3). The cell lysate of HEK293T/HaloMCM2 was mixed with the lysate of either HepG2/HisCore or HepG2/HisLacZ and incubated at 4 °C overnight. The bound fraction was washed and eluted with an elution buffer and analyzed by Western blotting. Lane 1, input of HepG2/HisCore; lane 2, input of HepG2/HisLacZ; lane 3, input of HEK293T/HaloMCM2; lane 4, HaloMCM2-bound fraction with HepG2/HisCore; and lane 5, HaloMCM2-bound fraction with HepG2/HisLacZ. “+” indicates the use of those listed to the right. HisCore and HisLacZ proteins were detected by an anti-His-tag antibody. HaloMCM2 was detected by an anti-Halo™-tag antibody. Endogenous MCMs were detected by specific antibodies, as described in Materials and Methods.

    Techniques Used: Pull Down Assay, Recombinant, Expressing, Incubation, Western Blot

    Immunofluorescence assay of HisCore and HaloMCMs in HEK293T cells. HisCore and individual HaloMCM were co-transfected into HEK293T cells. HisCore was detected by an anti-Core antibody (Red). HaloMCMs were detected by an anti-Halo™-tag antibody (Green). Nuclei were stained with DAPI (Blue).
    Figure Legend Snippet: Immunofluorescence assay of HisCore and HaloMCMs in HEK293T cells. HisCore and individual HaloMCM were co-transfected into HEK293T cells. HisCore was detected by an anti-Core antibody (Red). HaloMCMs were detected by an anti-Halo™-tag antibody (Green). Nuclei were stained with DAPI (Blue).

    Techniques Used: Immunofluorescence, Transfection, Staining

    hek 293t cells  (TaKaRa)


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    TaKaRa hek 293t cells
    Subcellular localization of Lc- SARM. (A) <t>HEK</t> <t>293T</t> cells were transfected with pTurbo-SARM-GFP and pTurboGFP-N (vector control), respectively. At 24 hpt, the cells were stained with DAPI and then detected and photographed under a confocal microscope. (B) The confirmation of the expression of Lc - SARM-GFP and pTurboGFP fusion proteins was conducted by Western blotting analysis using the Anti-TurboGFP antibody.
    Hek 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "SARM suppresses TRIF, TRAF3, and IRF3/7 mediated antiviral signaling in large yellow croaker Larimichthys crocea"

    Article Title: SARM suppresses TRIF, TRAF3, and IRF3/7 mediated antiviral signaling in large yellow croaker Larimichthys crocea

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2022.1021443

    Subcellular localization of Lc- SARM. (A) HEK 293T cells were transfected with pTurbo-SARM-GFP and pTurboGFP-N (vector control), respectively. At 24 hpt, the cells were stained with DAPI and then detected and photographed under a confocal microscope. (B) The confirmation of the expression of Lc - SARM-GFP and pTurboGFP fusion proteins was conducted by Western blotting analysis using the Anti-TurboGFP antibody.
    Figure Legend Snippet: Subcellular localization of Lc- SARM. (A) HEK 293T cells were transfected with pTurbo-SARM-GFP and pTurboGFP-N (vector control), respectively. At 24 hpt, the cells were stained with DAPI and then detected and photographed under a confocal microscope. (B) The confirmation of the expression of Lc - SARM-GFP and pTurboGFP fusion proteins was conducted by Western blotting analysis using the Anti-TurboGFP antibody.

    Techniques Used: Transfection, Plasmid Preparation, Staining, Microscopy, Expressing, Western Blot

    The role of Lc- SARM in the regulation of NF-κB signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) , and Lc- IRF7 (D) in the NF-κB promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates (1 × 10 5 cells/well) were co-transfected with 100 ng of pNF-κB-luc and 10 ng of pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two. For each transfection, the amounts of transfected plasmids were balanced with the pcDNA3.1 empty vector. At 24 hpt, the cells were collected and lysed for the measurement of luciferase activities. All data are shown as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.
    Figure Legend Snippet: The role of Lc- SARM in the regulation of NF-κB signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) , and Lc- IRF7 (D) in the NF-κB promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates (1 × 10 5 cells/well) were co-transfected with 100 ng of pNF-κB-luc and 10 ng of pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two. For each transfection, the amounts of transfected plasmids were balanced with the pcDNA3.1 empty vector. At 24 hpt, the cells were collected and lysed for the measurement of luciferase activities. All data are shown as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Techniques Used: Activation Assay, Luciferase, Transfection, Plasmid Preparation

    The role of Lc- SARM in the regulation of IRF3 signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the IRF3 promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates (1 × 10 5 cells/well) were co-transfected with 100 ng of pGL4-IRF3-pro and 10 ng of pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. The cells were harvested at 24 hpt and used for the measurement of luciferase activities. All data are expressed as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.
    Figure Legend Snippet: The role of Lc- SARM in the regulation of IRF3 signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the IRF3 promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates (1 × 10 5 cells/well) were co-transfected with 100 ng of pGL4-IRF3-pro and 10 ng of pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. The cells were harvested at 24 hpt and used for the measurement of luciferase activities. All data are expressed as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Techniques Used: Activation Assay, Luciferase, Transfection

    The role of Lc- SARM in the regulation of IRF7 signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the IRF7 promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates were co-transfected with 100 ng of pGL4-IRF7-pro and 10 ng pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. The cells were harvested at 24 hpt and used for the measurement of luciferase activities. All data are presented as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.
    Figure Legend Snippet: The role of Lc- SARM in the regulation of IRF7 signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the IRF7 promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates were co-transfected with 100 ng of pGL4-IRF7-pro and 10 ng pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. The cells were harvested at 24 hpt and used for the measurement of luciferase activities. All data are presented as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Techniques Used: Activation Assay, Luciferase, Transfection

    The role of Lc- SARM in the regulation of type I IFN signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the type I IFN promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates were co-transfected with 100 ng of pGL4-IFN1-pro and 10 ng pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. At 24 h post transfection, the cells were harvested and used for the detection of luciferase activities. All data are presented as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.
    Figure Legend Snippet: The role of Lc- SARM in the regulation of type I IFN signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the type I IFN promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates were co-transfected with 100 ng of pGL4-IFN1-pro and 10 ng pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. At 24 h post transfection, the cells were harvested and used for the detection of luciferase activities. All data are presented as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Techniques Used: Activation Assay, Luciferase, Transfection

    Interaction of Lc -SARM with Lc -TRIF, Lc -TRAF3, Lc -IRF3, and Lc -IRF7. HEK 293T cells seeded in 6-well plates (1 × 10 6 cells/well) were co-transfected with 2.5 μg of p3xFLAG-SARM together with 2.5 μg of pTurbo-TRIF-GFP, pTurbo-TRAF3-GFP, pTurbo-IRF3-GFP, pTurbo-IRF7-GFP, or pTurboGFP-N (vector control) in a combination of two. At 24 hpt, the cells were collected and lysed, and the cell lysates were immunoprecipitated with Anti-Flag antibody (covalently conjugated to agarose beads) and immunoblotting with Anti-TurboGFP antibody (upper panels). The Lc -SARM-Flag bound to Anti-Flag-agarose beads was shown by immunoblotting with Anti-Flag antibody (middle panels), and the cells lysates were also detected by immunoblotting with Anti-TurboGFP antibody (bottom panels), respectively.
    Figure Legend Snippet: Interaction of Lc -SARM with Lc -TRIF, Lc -TRAF3, Lc -IRF3, and Lc -IRF7. HEK 293T cells seeded in 6-well plates (1 × 10 6 cells/well) were co-transfected with 2.5 μg of p3xFLAG-SARM together with 2.5 μg of pTurbo-TRIF-GFP, pTurbo-TRAF3-GFP, pTurbo-IRF3-GFP, pTurbo-IRF7-GFP, or pTurboGFP-N (vector control) in a combination of two. At 24 hpt, the cells were collected and lysed, and the cell lysates were immunoprecipitated with Anti-Flag antibody (covalently conjugated to agarose beads) and immunoblotting with Anti-TurboGFP antibody (upper panels). The Lc -SARM-Flag bound to Anti-Flag-agarose beads was shown by immunoblotting with Anti-Flag antibody (middle panels), and the cells lysates were also detected by immunoblotting with Anti-TurboGFP antibody (bottom panels), respectively.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

    hek 293t cells  (TaKaRa)


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    TaKaRa hek 293t cells
    Hek 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hek293t cells  (TaKaRa)


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    TaKaRa hek293t cells
    ( A ) Schematic of caspase-4 dimerisation with the DmrB system, which allows controlled dimerisation by AP20187 (AP). All DmrB constructs were N-terminally V5-tagged. ( B ) Schematic of band sizes generated by cleavage at D270 and D289. These putative cleavage sites were mutated (D→A) to prevent auto-processing. An IDL uncl construct encoding mutations at both D270 and D289 sites was also included. ( C ) Caspase-4 activity was measured by relative fluorescence (RFU) generated by Ac-WEHD-AFC substrate cleavage. Caspase-4 was expressed in <t>HEK293T</t> cells and incubated with AP for 30 min prior to Ac-WEHD-AFC cleavage experiment. Linear regression analysis ( D ) showing rate of Ac-WEHD-AFC cleavage. Data are mean ± SEM of three biological replicates. Each data point represents an individual donor. p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****). ( E ) Caspase-4 constructs were transfected in HEK293T cells and dimerised by AP. Auto-processing was analysed by western blot of cell extracts.
    Hek293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Caspase-4 dimerisation and D289 auto-processing elicits an interleukin-1β converting enzyme"

    Article Title: Caspase-4 dimerisation and D289 auto-processing elicits an interleukin-1β converting enzyme

    Journal: bioRxiv

    doi: 10.1101/2023.01.05.522955

    ( A ) Schematic of caspase-4 dimerisation with the DmrB system, which allows controlled dimerisation by AP20187 (AP). All DmrB constructs were N-terminally V5-tagged. ( B ) Schematic of band sizes generated by cleavage at D270 and D289. These putative cleavage sites were mutated (D→A) to prevent auto-processing. An IDL uncl construct encoding mutations at both D270 and D289 sites was also included. ( C ) Caspase-4 activity was measured by relative fluorescence (RFU) generated by Ac-WEHD-AFC substrate cleavage. Caspase-4 was expressed in HEK293T cells and incubated with AP for 30 min prior to Ac-WEHD-AFC cleavage experiment. Linear regression analysis ( D ) showing rate of Ac-WEHD-AFC cleavage. Data are mean ± SEM of three biological replicates. Each data point represents an individual donor. p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****). ( E ) Caspase-4 constructs were transfected in HEK293T cells and dimerised by AP. Auto-processing was analysed by western blot of cell extracts.
    Figure Legend Snippet: ( A ) Schematic of caspase-4 dimerisation with the DmrB system, which allows controlled dimerisation by AP20187 (AP). All DmrB constructs were N-terminally V5-tagged. ( B ) Schematic of band sizes generated by cleavage at D270 and D289. These putative cleavage sites were mutated (D→A) to prevent auto-processing. An IDL uncl construct encoding mutations at both D270 and D289 sites was also included. ( C ) Caspase-4 activity was measured by relative fluorescence (RFU) generated by Ac-WEHD-AFC substrate cleavage. Caspase-4 was expressed in HEK293T cells and incubated with AP for 30 min prior to Ac-WEHD-AFC cleavage experiment. Linear regression analysis ( D ) showing rate of Ac-WEHD-AFC cleavage. Data are mean ± SEM of three biological replicates. Each data point represents an individual donor. p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****). ( E ) Caspase-4 constructs were transfected in HEK293T cells and dimerised by AP. Auto-processing was analysed by western blot of cell extracts.

    Techniques Used: Construct, Generated, Activity Assay, Fluorescence, Incubation, Transfection, Western Blot

    ( A - B ) Caspase-4 constructs were co-expressed with ( A ) V5-GSDMD or ( B ) pro-IL-1β in HEK293T cells and caspase-4 was dimerised by cell exposure to AP20187 (AP). Substrate cleavage was analysed by western blot of the whole cell lysates. ( C ) Recombinant caspase-4 or caspase-1 was incubated with recombinant human pro-IL-1β for 0 h, 6 h, and 24 h. All data ( A - C ) are representative of three biological replicates.
    Figure Legend Snippet: ( A - B ) Caspase-4 constructs were co-expressed with ( A ) V5-GSDMD or ( B ) pro-IL-1β in HEK293T cells and caspase-4 was dimerised by cell exposure to AP20187 (AP). Substrate cleavage was analysed by western blot of the whole cell lysates. ( C ) Recombinant caspase-4 or caspase-1 was incubated with recombinant human pro-IL-1β for 0 h, 6 h, and 24 h. All data ( A - C ) are representative of three biological replicates.

    Techniques Used: Construct, Western Blot, Recombinant, Incubation

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    ( A – C ) Barcode-specific gRNA-dependent activation of EGFP reporters for two barcoded <t>HEK293T</t> strains established for each of CloneSelect C→T ( A ), low-copy CRISPRa ( B ), and high-copy CRISPRa ( C ) (n=3). Scale bar, 50 μm. Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. ( D ) Median EGFP intensities of genome editing-activated EGFP positive cells (n=3). The Mann-Whitney U test was performed to compare two groups. ( E ) Comparison of Target-AID variants and a nCas9 (D10A) control in the CloneSelect C→T reporter activation for the same set of barcode-gRNA pairs (n=1). Welch’s t-test was performed to compare OT and NT activations. ( F ) Reporter activation in HeLa cells by CloneSelect C→T (n=3). Welch’s t-test was performed to compare OT and NT activations. Scale bar, 80 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.
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    Subcellular localization of Lc- SARM. (A) <t>HEK</t> <t>293T</t> cells were transfected with pTurbo-SARM-GFP and pTurboGFP-N (vector control), respectively. At 24 hpt, the cells were stained with DAPI and then detected and photographed under a confocal microscope. (B) The confirmation of the expression of Lc - SARM-GFP and pTurboGFP fusion proteins was conducted by Western blotting analysis using the Anti-TurboGFP antibody.
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    Subcellular localization of Lc- SARM. (A) <t>HEK</t> <t>293T</t> cells were transfected with pTurbo-SARM-GFP and pTurboGFP-N (vector control), respectively. At 24 hpt, the cells were stained with DAPI and then detected and photographed under a confocal microscope. (B) The confirmation of the expression of Lc - SARM-GFP and pTurboGFP fusion proteins was conducted by Western blotting analysis using the Anti-TurboGFP antibody.
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    ( A – C ) Barcode-specific gRNA-dependent activation of EGFP reporters for two barcoded HEK293T strains established for each of CloneSelect C→T ( A ), low-copy CRISPRa ( B ), and high-copy CRISPRa ( C ) (n=3). Scale bar, 50 μm. Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. ( D ) Median EGFP intensities of genome editing-activated EGFP positive cells (n=3). The Mann-Whitney U test was performed to compare two groups. ( E ) Comparison of Target-AID variants and a nCas9 (D10A) control in the CloneSelect C→T reporter activation for the same set of barcode-gRNA pairs (n=1). Welch’s t-test was performed to compare OT and NT activations. ( F ) Reporter activation in HeLa cells by CloneSelect C→T (n=3). Welch’s t-test was performed to compare OT and NT activations. Scale bar, 80 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.

    Journal: bioRxiv

    Article Title: A multi-kingdom genetic barcoding system for precise target clone isolation

    doi: 10.1101/2023.01.18.524633

    Figure Lengend Snippet: ( A – C ) Barcode-specific gRNA-dependent activation of EGFP reporters for two barcoded HEK293T strains established for each of CloneSelect C→T ( A ), low-copy CRISPRa ( B ), and high-copy CRISPRa ( C ) (n=3). Scale bar, 50 μm. Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. ( D ) Median EGFP intensities of genome editing-activated EGFP positive cells (n=3). The Mann-Whitney U test was performed to compare two groups. ( E ) Comparison of Target-AID variants and a nCas9 (D10A) control in the CloneSelect C→T reporter activation for the same set of barcode-gRNA pairs (n=1). Welch’s t-test was performed to compare OT and NT activations. ( F ) Reporter activation in HeLa cells by CloneSelect C→T (n=3). Welch’s t-test was performed to compare OT and NT activations. Scale bar, 80 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.

    Article Snippet: Human embryonic kidney 293Ta (HEK293Ta) and HEK293T Lenti-X cells were purchased from GeneCopoeia (#LT008) and Takara (#632180), respectively.

    Techniques: Activation Assay, MANN-WHITNEY

    ( A ) Barcode-specific gRNA-dependent reporter activation circuit using wild-type Cas9. ( B ) Barcode-specific activation of the deletion-based reporter prepared for two barcodes (BC-del1 and BC-del2) in HEK293T cells (n=3). Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 50 μm. ( C ) ROC curves along varying reporter intensity thresholds for target barcoded cells (n=3).

    Journal: bioRxiv

    Article Title: A multi-kingdom genetic barcoding system for precise target clone isolation

    doi: 10.1101/2023.01.18.524633

    Figure Lengend Snippet: ( A ) Barcode-specific gRNA-dependent reporter activation circuit using wild-type Cas9. ( B ) Barcode-specific activation of the deletion-based reporter prepared for two barcodes (BC-del1 and BC-del2) in HEK293T cells (n=3). Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 50 μm. ( C ) ROC curves along varying reporter intensity thresholds for target barcoded cells (n=3).

    Article Snippet: Human embryonic kidney 293Ta (HEK293Ta) and HEK293T Lenti-X cells were purchased from GeneCopoeia (#LT008) and Takara (#632180), respectively.

    Techniques: Activation Assay

    ( A ) scCloneSelect. ( B and C ) Barcode-specific gRNA-dependent reporter activation of the original CloneSelect C→T and scCloneSelect in HEK293T cells (n=3). Scale bar, 50 μm. ( D and E ) Barcode-specific gRNA-dependent reporter activation of three barcoded mESC lines by scCloneSelect. Target-AID was stably integrated prior to the barcoding. gRNAs were delivered by lentiviral transduction. Scale bar, 100 μm. Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. * P < 0.05; ** P < 0.01; *** P < 0.001. ( F ) Schematic diagram of a scCloneSelect workflow to retrospectively isolate a cell clone demonstrating a gene expression profile of interest from a cell population stored before they demonstrate the target gene expression pattern. ( G ) mESC cell culture assays and clone isolation performed in this work. ( H ) scRNA-seq of mESC populations treated with LIF and 2i and those without LIF or 2i. ( I ) Distribution of cells for arbitrarily selected clones in the two-dimensional embedding of high-dimensional gene expression space by UMAP (uniform manifold approximation and projection). ( J ) Abundance of barcoded cell clones in the mESC population. The data was generated based on dntags identified by reamplifying the dntag reads from the original scRNA-seq libraries. ( K ) gRNA-specific activation of target barcoded clones in the mESC population. Scale bar, 50 μm. ( L ) Barcode enrichment analysis after cell sorting of the reporter-activated cells. Each row represents the barcode enrichment profile for each target isolation assay. The left heatmap was expanded from the dashed box area of the right heatmap.

    Journal: bioRxiv

    Article Title: A multi-kingdom genetic barcoding system for precise target clone isolation

    doi: 10.1101/2023.01.18.524633

    Figure Lengend Snippet: ( A ) scCloneSelect. ( B and C ) Barcode-specific gRNA-dependent reporter activation of the original CloneSelect C→T and scCloneSelect in HEK293T cells (n=3). Scale bar, 50 μm. ( D and E ) Barcode-specific gRNA-dependent reporter activation of three barcoded mESC lines by scCloneSelect. Target-AID was stably integrated prior to the barcoding. gRNAs were delivered by lentiviral transduction. Scale bar, 100 μm. Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations. * P < 0.05; ** P < 0.01; *** P < 0.001. ( F ) Schematic diagram of a scCloneSelect workflow to retrospectively isolate a cell clone demonstrating a gene expression profile of interest from a cell population stored before they demonstrate the target gene expression pattern. ( G ) mESC cell culture assays and clone isolation performed in this work. ( H ) scRNA-seq of mESC populations treated with LIF and 2i and those without LIF or 2i. ( I ) Distribution of cells for arbitrarily selected clones in the two-dimensional embedding of high-dimensional gene expression space by UMAP (uniform manifold approximation and projection). ( J ) Abundance of barcoded cell clones in the mESC population. The data was generated based on dntags identified by reamplifying the dntag reads from the original scRNA-seq libraries. ( K ) gRNA-specific activation of target barcoded clones in the mESC population. Scale bar, 50 μm. ( L ) Barcode enrichment analysis after cell sorting of the reporter-activated cells. Each row represents the barcode enrichment profile for each target isolation assay. The left heatmap was expanded from the dashed box area of the right heatmap.

    Article Snippet: Human embryonic kidney 293Ta (HEK293Ta) and HEK293T Lenti-X cells were purchased from GeneCopoeia (#LT008) and Takara (#632180), respectively.

    Techniques: Activation Assay, Stable Transfection, Transduction, Expressing, Cell Culture, Isolation, Clone Assay, Generated, FACS

    ( A ) EGFP-positive control expressions for the original CloneSelect C→T and scCloneSelect in HEK293T cells with the same genome editing conditions tested for the respective reporters (n=3). Scale bar, 50 μm. ( B ) Median EGFP intensities of base editing-activated EGFP positive cells (n=3). ( C and D ) Barcode-specific gRNA-dependent reporter activation of six barcoded cell lines by scCloneSelect (n=1). Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 50 μm. ( E ) RT-PCR of the scCloneSelect dntags in HEK293T. ( F ) Fraction of mESC single-cell transcriptome profiles (Drop-seq) that contained dntags and fraction of dntags reported in the uptag-dntag combination reference database. ( G ) Schematic representation of a scCloneSelect reporter activation assay where Target-AID was stably introduced to the cell population prior to barcoding and gRNA-dependent reporter activation. ( H and I ) gRNA-dependent reporter activation of target barcoded mESCs and CA1 hPSCs by scCloneSelect (n=2). Target-AID was stably integrated prior to the barcoding. Targeting gRNAs were delivered by transfection. Welch’s t-test was performed to compare OT and NT activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 100 μm. ( J ) Schematic representation of a scCloneSelect reporter activation assay where the target gRNA and Target-AID were electroporated together to the barcoded cell population. ( K ) gRNA-dependent reporter activation of barcoded H1 hPSCs by scCloneSelect (n=2). Targeting gRNA and Target-AID were electroporated together. Scale bar, 100 μm.

    Journal: bioRxiv

    Article Title: A multi-kingdom genetic barcoding system for precise target clone isolation

    doi: 10.1101/2023.01.18.524633

    Figure Lengend Snippet: ( A ) EGFP-positive control expressions for the original CloneSelect C→T and scCloneSelect in HEK293T cells with the same genome editing conditions tested for the respective reporters (n=3). Scale bar, 50 μm. ( B ) Median EGFP intensities of base editing-activated EGFP positive cells (n=3). ( C and D ) Barcode-specific gRNA-dependent reporter activation of six barcoded cell lines by scCloneSelect (n=1). Welch’s t-test was performed to compare on-target (OT) and non-target (NT) activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 50 μm. ( E ) RT-PCR of the scCloneSelect dntags in HEK293T. ( F ) Fraction of mESC single-cell transcriptome profiles (Drop-seq) that contained dntags and fraction of dntags reported in the uptag-dntag combination reference database. ( G ) Schematic representation of a scCloneSelect reporter activation assay where Target-AID was stably introduced to the cell population prior to barcoding and gRNA-dependent reporter activation. ( H and I ) gRNA-dependent reporter activation of target barcoded mESCs and CA1 hPSCs by scCloneSelect (n=2). Target-AID was stably integrated prior to the barcoding. Targeting gRNAs were delivered by transfection. Welch’s t-test was performed to compare OT and NT activations (* P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar, 100 μm. ( J ) Schematic representation of a scCloneSelect reporter activation assay where the target gRNA and Target-AID were electroporated together to the barcoded cell population. ( K ) gRNA-dependent reporter activation of barcoded H1 hPSCs by scCloneSelect (n=2). Targeting gRNA and Target-AID were electroporated together. Scale bar, 100 μm.

    Article Snippet: Human embryonic kidney 293Ta (HEK293Ta) and HEK293T Lenti-X cells were purchased from GeneCopoeia (#LT008) and Takara (#632180), respectively.

    Techniques: Positive Control, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection

    Subcellular localization of Lc- SARM. (A) HEK 293T cells were transfected with pTurbo-SARM-GFP and pTurboGFP-N (vector control), respectively. At 24 hpt, the cells were stained with DAPI and then detected and photographed under a confocal microscope. (B) The confirmation of the expression of Lc - SARM-GFP and pTurboGFP fusion proteins was conducted by Western blotting analysis using the Anti-TurboGFP antibody.

    Journal: Frontiers in Immunology

    Article Title: SARM suppresses TRIF, TRAF3, and IRF3/7 mediated antiviral signaling in large yellow croaker Larimichthys crocea

    doi: 10.3389/fimmu.2022.1021443

    Figure Lengend Snippet: Subcellular localization of Lc- SARM. (A) HEK 293T cells were transfected with pTurbo-SARM-GFP and pTurboGFP-N (vector control), respectively. At 24 hpt, the cells were stained with DAPI and then detected and photographed under a confocal microscope. (B) The confirmation of the expression of Lc - SARM-GFP and pTurboGFP fusion proteins was conducted by Western blotting analysis using the Anti-TurboGFP antibody.

    Article Snippet: To understand the association of large yellow croaker SARM and TRIF, TRAF3, IRF3, or IRF7, HEK 293T cells in 24-well plates (1 × 10 5 cells/well) were transiently co-transfected with pNF-κB-luc (100 ng/well, Clontech, Palo Alto, CA), pGL4-IRF3-pro (100 ng/well, Chinese invention patent number: ZL201710457836.8), pGL4-IRF7-pro (100 ng/well, Chinese invention patent number: ZL201710457820.7), or pGL4-IFN1-pro (Chinese invention patent application number: 201710456729.3), and pRL-TK (10 ng/well, Promega, Madison, WI) together with 100 ng pcDNA3.1-SARM and pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, or pcDNA3.1-IRF7 alone or in a combination of two using Lipofectamine 3000.

    Techniques: Transfection, Plasmid Preparation, Staining, Microscopy, Expressing, Western Blot

    The role of Lc- SARM in the regulation of NF-κB signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) , and Lc- IRF7 (D) in the NF-κB promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates (1 × 10 5 cells/well) were co-transfected with 100 ng of pNF-κB-luc and 10 ng of pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two. For each transfection, the amounts of transfected plasmids were balanced with the pcDNA3.1 empty vector. At 24 hpt, the cells were collected and lysed for the measurement of luciferase activities. All data are shown as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Journal: Frontiers in Immunology

    Article Title: SARM suppresses TRIF, TRAF3, and IRF3/7 mediated antiviral signaling in large yellow croaker Larimichthys crocea

    doi: 10.3389/fimmu.2022.1021443

    Figure Lengend Snippet: The role of Lc- SARM in the regulation of NF-κB signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) , and Lc- IRF7 (D) in the NF-κB promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates (1 × 10 5 cells/well) were co-transfected with 100 ng of pNF-κB-luc and 10 ng of pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two. For each transfection, the amounts of transfected plasmids were balanced with the pcDNA3.1 empty vector. At 24 hpt, the cells were collected and lysed for the measurement of luciferase activities. All data are shown as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Article Snippet: To understand the association of large yellow croaker SARM and TRIF, TRAF3, IRF3, or IRF7, HEK 293T cells in 24-well plates (1 × 10 5 cells/well) were transiently co-transfected with pNF-κB-luc (100 ng/well, Clontech, Palo Alto, CA), pGL4-IRF3-pro (100 ng/well, Chinese invention patent number: ZL201710457836.8), pGL4-IRF7-pro (100 ng/well, Chinese invention patent number: ZL201710457820.7), or pGL4-IFN1-pro (Chinese invention patent application number: 201710456729.3), and pRL-TK (10 ng/well, Promega, Madison, WI) together with 100 ng pcDNA3.1-SARM and pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, or pcDNA3.1-IRF7 alone or in a combination of two using Lipofectamine 3000.

    Techniques: Activation Assay, Luciferase, Transfection, Plasmid Preparation

    The role of Lc- SARM in the regulation of IRF3 signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the IRF3 promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates (1 × 10 5 cells/well) were co-transfected with 100 ng of pGL4-IRF3-pro and 10 ng of pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. The cells were harvested at 24 hpt and used for the measurement of luciferase activities. All data are expressed as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Journal: Frontiers in Immunology

    Article Title: SARM suppresses TRIF, TRAF3, and IRF3/7 mediated antiviral signaling in large yellow croaker Larimichthys crocea

    doi: 10.3389/fimmu.2022.1021443

    Figure Lengend Snippet: The role of Lc- SARM in the regulation of IRF3 signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the IRF3 promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates (1 × 10 5 cells/well) were co-transfected with 100 ng of pGL4-IRF3-pro and 10 ng of pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. The cells were harvested at 24 hpt and used for the measurement of luciferase activities. All data are expressed as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Article Snippet: To understand the association of large yellow croaker SARM and TRIF, TRAF3, IRF3, or IRF7, HEK 293T cells in 24-well plates (1 × 10 5 cells/well) were transiently co-transfected with pNF-κB-luc (100 ng/well, Clontech, Palo Alto, CA), pGL4-IRF3-pro (100 ng/well, Chinese invention patent number: ZL201710457836.8), pGL4-IRF7-pro (100 ng/well, Chinese invention patent number: ZL201710457820.7), or pGL4-IFN1-pro (Chinese invention patent application number: 201710456729.3), and pRL-TK (10 ng/well, Promega, Madison, WI) together with 100 ng pcDNA3.1-SARM and pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, or pcDNA3.1-IRF7 alone or in a combination of two using Lipofectamine 3000.

    Techniques: Activation Assay, Luciferase, Transfection

    The role of Lc- SARM in the regulation of IRF7 signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the IRF7 promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates were co-transfected with 100 ng of pGL4-IRF7-pro and 10 ng pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. The cells were harvested at 24 hpt and used for the measurement of luciferase activities. All data are presented as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Journal: Frontiers in Immunology

    Article Title: SARM suppresses TRIF, TRAF3, and IRF3/7 mediated antiviral signaling in large yellow croaker Larimichthys crocea

    doi: 10.3389/fimmu.2022.1021443

    Figure Lengend Snippet: The role of Lc- SARM in the regulation of IRF7 signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the IRF7 promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates were co-transfected with 100 ng of pGL4-IRF7-pro and 10 ng pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. The cells were harvested at 24 hpt and used for the measurement of luciferase activities. All data are presented as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Article Snippet: To understand the association of large yellow croaker SARM and TRIF, TRAF3, IRF3, or IRF7, HEK 293T cells in 24-well plates (1 × 10 5 cells/well) were transiently co-transfected with pNF-κB-luc (100 ng/well, Clontech, Palo Alto, CA), pGL4-IRF3-pro (100 ng/well, Chinese invention patent number: ZL201710457836.8), pGL4-IRF7-pro (100 ng/well, Chinese invention patent number: ZL201710457820.7), or pGL4-IFN1-pro (Chinese invention patent application number: 201710456729.3), and pRL-TK (10 ng/well, Promega, Madison, WI) together with 100 ng pcDNA3.1-SARM and pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, or pcDNA3.1-IRF7 alone or in a combination of two using Lipofectamine 3000.

    Techniques: Activation Assay, Luciferase, Transfection

    The role of Lc- SARM in the regulation of type I IFN signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the type I IFN promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates were co-transfected with 100 ng of pGL4-IFN1-pro and 10 ng pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. At 24 h post transfection, the cells were harvested and used for the detection of luciferase activities. All data are presented as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Journal: Frontiers in Immunology

    Article Title: SARM suppresses TRIF, TRAF3, and IRF3/7 mediated antiviral signaling in large yellow croaker Larimichthys crocea

    doi: 10.3389/fimmu.2022.1021443

    Figure Lengend Snippet: The role of Lc- SARM in the regulation of type I IFN signaling. The associations of Lc- SARM with Lc- TRIF (A) , Lc- TRAF3 (B) , Lc- IRF3 (C) and Lc- IRF7 (D) in the type I IFN promoter activation were analyzed by luciferase reporter assays. HEK 293T cells seeded in 24-well plates were co-transfected with 100 ng of pGL4-IFN1-pro and 10 ng pRL-TK together with 100 ng of pcDNA3.1-SARM, pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, and pcDNA3.1-IRF7 alone or in a combination of two as described above. At 24 h post transfection, the cells were harvested and used for the detection of luciferase activities. All data are presented as mean of three independent experiments, with bars representing the SE. * P < 0.05; ** P < 0.01; NS, not significant.

    Article Snippet: To understand the association of large yellow croaker SARM and TRIF, TRAF3, IRF3, or IRF7, HEK 293T cells in 24-well plates (1 × 10 5 cells/well) were transiently co-transfected with pNF-κB-luc (100 ng/well, Clontech, Palo Alto, CA), pGL4-IRF3-pro (100 ng/well, Chinese invention patent number: ZL201710457836.8), pGL4-IRF7-pro (100 ng/well, Chinese invention patent number: ZL201710457820.7), or pGL4-IFN1-pro (Chinese invention patent application number: 201710456729.3), and pRL-TK (10 ng/well, Promega, Madison, WI) together with 100 ng pcDNA3.1-SARM and pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, or pcDNA3.1-IRF7 alone or in a combination of two using Lipofectamine 3000.

    Techniques: Activation Assay, Luciferase, Transfection

    Interaction of Lc -SARM with Lc -TRIF, Lc -TRAF3, Lc -IRF3, and Lc -IRF7. HEK 293T cells seeded in 6-well plates (1 × 10 6 cells/well) were co-transfected with 2.5 μg of p3xFLAG-SARM together with 2.5 μg of pTurbo-TRIF-GFP, pTurbo-TRAF3-GFP, pTurbo-IRF3-GFP, pTurbo-IRF7-GFP, or pTurboGFP-N (vector control) in a combination of two. At 24 hpt, the cells were collected and lysed, and the cell lysates were immunoprecipitated with Anti-Flag antibody (covalently conjugated to agarose beads) and immunoblotting with Anti-TurboGFP antibody (upper panels). The Lc -SARM-Flag bound to Anti-Flag-agarose beads was shown by immunoblotting with Anti-Flag antibody (middle panels), and the cells lysates were also detected by immunoblotting with Anti-TurboGFP antibody (bottom panels), respectively.

    Journal: Frontiers in Immunology

    Article Title: SARM suppresses TRIF, TRAF3, and IRF3/7 mediated antiviral signaling in large yellow croaker Larimichthys crocea

    doi: 10.3389/fimmu.2022.1021443

    Figure Lengend Snippet: Interaction of Lc -SARM with Lc -TRIF, Lc -TRAF3, Lc -IRF3, and Lc -IRF7. HEK 293T cells seeded in 6-well plates (1 × 10 6 cells/well) were co-transfected with 2.5 μg of p3xFLAG-SARM together with 2.5 μg of pTurbo-TRIF-GFP, pTurbo-TRAF3-GFP, pTurbo-IRF3-GFP, pTurbo-IRF7-GFP, or pTurboGFP-N (vector control) in a combination of two. At 24 hpt, the cells were collected and lysed, and the cell lysates were immunoprecipitated with Anti-Flag antibody (covalently conjugated to agarose beads) and immunoblotting with Anti-TurboGFP antibody (upper panels). The Lc -SARM-Flag bound to Anti-Flag-agarose beads was shown by immunoblotting with Anti-Flag antibody (middle panels), and the cells lysates were also detected by immunoblotting with Anti-TurboGFP antibody (bottom panels), respectively.

    Article Snippet: To understand the association of large yellow croaker SARM and TRIF, TRAF3, IRF3, or IRF7, HEK 293T cells in 24-well plates (1 × 10 5 cells/well) were transiently co-transfected with pNF-κB-luc (100 ng/well, Clontech, Palo Alto, CA), pGL4-IRF3-pro (100 ng/well, Chinese invention patent number: ZL201710457836.8), pGL4-IRF7-pro (100 ng/well, Chinese invention patent number: ZL201710457820.7), or pGL4-IFN1-pro (Chinese invention patent application number: 201710456729.3), and pRL-TK (10 ng/well, Promega, Madison, WI) together with 100 ng pcDNA3.1-SARM and pcDNA3.1-TRIF, pcDNA3.1-TRAF3, pcDNA3.1-IRF3, or pcDNA3.1-IRF7 alone or in a combination of two using Lipofectamine 3000.

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot