calcium phosphate co transfection  (ATCC)


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    ATCC calcium phosphate co transfection
    ( A ) Illustration of a generic integral membrane protein (representing SYT1 and SYB2) with a luminal HaloTag to allow for selective labeling at the PM. ( B ) Schematic of the HaloTag ligand (HTL) labeling protocol, shown within a nerve terminal, with the non-permeant ligand incubation step to label surface protein. If SYT1 and SYB2 are first delivered to an internal sorting compartment ( i ), rather than the PM ( ii ) prior to SV or SV-intermediate formation, they will not pass through the PM and so will not be labeled by the non-permeant ligand (green). Incubation with permeant ligand labels the remaining tagged protein and the resulting change in fluorescence denotes the efficiency of PM delivery. The signal from labeling with the non-permeant ligand was referred to as F initial , where the unlabeled control coverslips still yielded a small background signal, producing a reproducible non-zero value that allowed us to calculate ratios. The subsequent signal after labeling with permeant ligand was called F final . This labeling step included unbound ligand which, while weak and diffuse, results in a slight increase in the background signal. To counteract this, ROIs were drawn to include only the fluorescence intensity within the synapse. ( C ) Timeline for the <t>transfection</t> and labeling protocols. Briefly, cultured rat hippocampal neurons were transduced with TeTx-LC virus on 5 days in vitro (DIV) and then co-transfected on 9 DIV with HaloTag-SYT1 or SYB2-HaloTag, and SYP-GFP; the GFP construct was included to mark synapses and ‘dilute’ the HaloTag plasmid to achieve lower expression levels. Half of the coverslips were incubated in non-permeant HTL (JF549i) immediately after co-transfection to label any tagged protein that was delivered to the PM. Six days later (15 DIV) neurons were rinsed, imaged, and incubated with permeant ligand (JF549), to label any remaining tagged protein, and imaged again. ( D ) Immunoblot of cells transduced with a virus expressing TeTx-LC, resulting in the cleavage of endogenous SYB2 and the inhibition of SV recycling. We note that the SYB2 fusion protein used in these experiments harbored two point mutations to render it resistant to TeTx-LC (see Methods). Blots were probed for endogenous SYB2, SYT1, and SYP, with a TCE loading control. The normalized (F final /F initial ) change in fluorescence intensity of the SYT1 ( E ) and SYB2 ( F ) fusion proteins upon adding permeant fluorescent ligand to cells grown with or without non-permeant ligand for 6 days; mean values with 95% CI are plotted to the right of each scatter plot. Data were analyzed with unpaired t-tests for both proteins; p-values = <0.0001. Panel ( E ) contains data from 156 synapses cultured in the presence of JF549i, and 136 synapses grown in the absence of this HTL. Data for both groups were from 8 fields of view from 4 different litters. Panel ( F ) contains data from 107 synapses cultured in the presence of JF549i, and 79 synapses grown in the absence of this HTL. Data for both groups were from five fields of view from three different litters. Mean values and descriptive statistics for SYT1 and SYB2 can be found in . Panels ( G, H ) are representative images of SYP-GFP to mark synapses (dashed circles), and the corresponding HaloTag-SYT1 signals under the indicated conditions; in the bottom panels the JF549 ligand was not washed away, resulting in a higher background. For all conditions, identical laser and gain settings were used. Any linear brightness and contrast adjustments were applied to all conditions. ( I, J ) Same as panels ( G ) and ( H ), but for SYB2-HaloTag. Figure 5—source data 1. Descriptive statistics corresponding to . (A) Corresponds to . (B) Corresponds to .
    Calcium Phosphate Co Transfection, supplied by ATCC, 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 "Synaptic vesicle proteins are selectively delivered to axons in mammalian neurons"

    Article Title: Synaptic vesicle proteins are selectively delivered to axons in mammalian neurons

    Journal: eLife

    doi: 10.7554/eLife.82568

    ( A ) Illustration of a generic integral membrane protein (representing SYT1 and SYB2) with a luminal HaloTag to allow for selective labeling at the PM. ( B ) Schematic of the HaloTag ligand (HTL) labeling protocol, shown within a nerve terminal, with the non-permeant ligand incubation step to label surface protein. If SYT1 and SYB2 are first delivered to an internal sorting compartment ( i ), rather than the PM ( ii ) prior to SV or SV-intermediate formation, they will not pass through the PM and so will not be labeled by the non-permeant ligand (green). Incubation with permeant ligand labels the remaining tagged protein and the resulting change in fluorescence denotes the efficiency of PM delivery. The signal from labeling with the non-permeant ligand was referred to as F initial , where the unlabeled control coverslips still yielded a small background signal, producing a reproducible non-zero value that allowed us to calculate ratios. The subsequent signal after labeling with permeant ligand was called F final . This labeling step included unbound ligand which, while weak and diffuse, results in a slight increase in the background signal. To counteract this, ROIs were drawn to include only the fluorescence intensity within the synapse. ( C ) Timeline for the transfection and labeling protocols. Briefly, cultured rat hippocampal neurons were transduced with TeTx-LC virus on 5 days in vitro (DIV) and then co-transfected on 9 DIV with HaloTag-SYT1 or SYB2-HaloTag, and SYP-GFP; the GFP construct was included to mark synapses and ‘dilute’ the HaloTag plasmid to achieve lower expression levels. Half of the coverslips were incubated in non-permeant HTL (JF549i) immediately after co-transfection to label any tagged protein that was delivered to the PM. Six days later (15 DIV) neurons were rinsed, imaged, and incubated with permeant ligand (JF549), to label any remaining tagged protein, and imaged again. ( D ) Immunoblot of cells transduced with a virus expressing TeTx-LC, resulting in the cleavage of endogenous SYB2 and the inhibition of SV recycling. We note that the SYB2 fusion protein used in these experiments harbored two point mutations to render it resistant to TeTx-LC (see Methods). Blots were probed for endogenous SYB2, SYT1, and SYP, with a TCE loading control. The normalized (F final /F initial ) change in fluorescence intensity of the SYT1 ( E ) and SYB2 ( F ) fusion proteins upon adding permeant fluorescent ligand to cells grown with or without non-permeant ligand for 6 days; mean values with 95% CI are plotted to the right of each scatter plot. Data were analyzed with unpaired t-tests for both proteins; p-values = <0.0001. Panel ( E ) contains data from 156 synapses cultured in the presence of JF549i, and 136 synapses grown in the absence of this HTL. Data for both groups were from 8 fields of view from 4 different litters. Panel ( F ) contains data from 107 synapses cultured in the presence of JF549i, and 79 synapses grown in the absence of this HTL. Data for both groups were from five fields of view from three different litters. Mean values and descriptive statistics for SYT1 and SYB2 can be found in . Panels ( G, H ) are representative images of SYP-GFP to mark synapses (dashed circles), and the corresponding HaloTag-SYT1 signals under the indicated conditions; in the bottom panels the JF549 ligand was not washed away, resulting in a higher background. For all conditions, identical laser and gain settings were used. Any linear brightness and contrast adjustments were applied to all conditions. ( I, J ) Same as panels ( G ) and ( H ), but for SYB2-HaloTag. Figure 5—source data 1. Descriptive statistics corresponding to . (A) Corresponds to . (B) Corresponds to .
    Figure Legend Snippet: ( A ) Illustration of a generic integral membrane protein (representing SYT1 and SYB2) with a luminal HaloTag to allow for selective labeling at the PM. ( B ) Schematic of the HaloTag ligand (HTL) labeling protocol, shown within a nerve terminal, with the non-permeant ligand incubation step to label surface protein. If SYT1 and SYB2 are first delivered to an internal sorting compartment ( i ), rather than the PM ( ii ) prior to SV or SV-intermediate formation, they will not pass through the PM and so will not be labeled by the non-permeant ligand (green). Incubation with permeant ligand labels the remaining tagged protein and the resulting change in fluorescence denotes the efficiency of PM delivery. The signal from labeling with the non-permeant ligand was referred to as F initial , where the unlabeled control coverslips still yielded a small background signal, producing a reproducible non-zero value that allowed us to calculate ratios. The subsequent signal after labeling with permeant ligand was called F final . This labeling step included unbound ligand which, while weak and diffuse, results in a slight increase in the background signal. To counteract this, ROIs were drawn to include only the fluorescence intensity within the synapse. ( C ) Timeline for the transfection and labeling protocols. Briefly, cultured rat hippocampal neurons were transduced with TeTx-LC virus on 5 days in vitro (DIV) and then co-transfected on 9 DIV with HaloTag-SYT1 or SYB2-HaloTag, and SYP-GFP; the GFP construct was included to mark synapses and ‘dilute’ the HaloTag plasmid to achieve lower expression levels. Half of the coverslips were incubated in non-permeant HTL (JF549i) immediately after co-transfection to label any tagged protein that was delivered to the PM. Six days later (15 DIV) neurons were rinsed, imaged, and incubated with permeant ligand (JF549), to label any remaining tagged protein, and imaged again. ( D ) Immunoblot of cells transduced with a virus expressing TeTx-LC, resulting in the cleavage of endogenous SYB2 and the inhibition of SV recycling. We note that the SYB2 fusion protein used in these experiments harbored two point mutations to render it resistant to TeTx-LC (see Methods). Blots were probed for endogenous SYB2, SYT1, and SYP, with a TCE loading control. The normalized (F final /F initial ) change in fluorescence intensity of the SYT1 ( E ) and SYB2 ( F ) fusion proteins upon adding permeant fluorescent ligand to cells grown with or without non-permeant ligand for 6 days; mean values with 95% CI are plotted to the right of each scatter plot. Data were analyzed with unpaired t-tests for both proteins; p-values = <0.0001. Panel ( E ) contains data from 156 synapses cultured in the presence of JF549i, and 136 synapses grown in the absence of this HTL. Data for both groups were from 8 fields of view from 4 different litters. Panel ( F ) contains data from 107 synapses cultured in the presence of JF549i, and 79 synapses grown in the absence of this HTL. Data for both groups were from five fields of view from three different litters. Mean values and descriptive statistics for SYT1 and SYB2 can be found in . Panels ( G, H ) are representative images of SYP-GFP to mark synapses (dashed circles), and the corresponding HaloTag-SYT1 signals under the indicated conditions; in the bottom panels the JF549 ligand was not washed away, resulting in a higher background. For all conditions, identical laser and gain settings were used. Any linear brightness and contrast adjustments were applied to all conditions. ( I, J ) Same as panels ( G ) and ( H ), but for SYB2-HaloTag. Figure 5—source data 1. Descriptive statistics corresponding to . (A) Corresponds to . (B) Corresponds to .

    Techniques Used: Labeling, Incubation, Fluorescence, Transfection, Cell Culture, Transduction, In Vitro, Construct, Plasmid Preparation, Expressing, Cotransfection, Western Blot, Inhibition

    sirna transfections  (ATCC)


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    ATCC sirna transfections
    Sirna Transfections, supplied by ATCC, 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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    transfex transfection reagent  (ATCC)


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    ATCC transfex transfection reagent
    Transfex Transfection Reagent, supplied by ATCC, 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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    polyfast transfection reagent  (ATCC)


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    ATCC polyfast transfection reagent
    Characterization of du-cGAS functional domains. DEFs cells were transfected with expression plasmids encoding full-length du-cGAS, truncated du-cGAS or empty vector (EV). After 24 h <t>post-transfection,</t> cells were infected with DAdV-B2 at the MOI of 1.0 and harvested at 48 hpi. (A) Cell monolayers were detected the expression of DAdV-B2 hexon protein by IFA. (a) empty plasmid; (b) du-cGAS; (c) du-cGAS (69-423aa) (d) Mab21; (e) Zinc-Ribbon; (f) NTase. (B) The viral titers in the cell culture supernatant were detected by TCID 50 assay. (C) The protein expression of full-length and truncated du-cGAS, and DAdV-B2 hexon was detected by western blotting. (D) The relative mRNA expression of type I IFNs, STING and key ISGs was detected by qRT-PCR. Data represent the mean values ± SD, and statistical significance was analyzed by t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.
    Polyfast Transfection Reagent, supplied by ATCC, 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 "Duck cGAS inhibits DNA and RNA virus replication by activating IFNs and antiviral ISGs"

    Article Title: Duck cGAS inhibits DNA and RNA virus replication by activating IFNs and antiviral ISGs

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2023.1101335

    Characterization of du-cGAS functional domains. DEFs cells were transfected with expression plasmids encoding full-length du-cGAS, truncated du-cGAS or empty vector (EV). After 24 h post-transfection, cells were infected with DAdV-B2 at the MOI of 1.0 and harvested at 48 hpi. (A) Cell monolayers were detected the expression of DAdV-B2 hexon protein by IFA. (a) empty plasmid; (b) du-cGAS; (c) du-cGAS (69-423aa) (d) Mab21; (e) Zinc-Ribbon; (f) NTase. (B) The viral titers in the cell culture supernatant were detected by TCID 50 assay. (C) The protein expression of full-length and truncated du-cGAS, and DAdV-B2 hexon was detected by western blotting. (D) The relative mRNA expression of type I IFNs, STING and key ISGs was detected by qRT-PCR. Data represent the mean values ± SD, and statistical significance was analyzed by t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.
    Figure Legend Snippet: Characterization of du-cGAS functional domains. DEFs cells were transfected with expression plasmids encoding full-length du-cGAS, truncated du-cGAS or empty vector (EV). After 24 h post-transfection, cells were infected with DAdV-B2 at the MOI of 1.0 and harvested at 48 hpi. (A) Cell monolayers were detected the expression of DAdV-B2 hexon protein by IFA. (a) empty plasmid; (b) du-cGAS; (c) du-cGAS (69-423aa) (d) Mab21; (e) Zinc-Ribbon; (f) NTase. (B) The viral titers in the cell culture supernatant were detected by TCID 50 assay. (C) The protein expression of full-length and truncated du-cGAS, and DAdV-B2 hexon was detected by western blotting. (D) The relative mRNA expression of type I IFNs, STING and key ISGs was detected by qRT-PCR. Data represent the mean values ± SD, and statistical significance was analyzed by t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.

    Techniques Used: Functional Assay, Transfection, Expressing, Plasmid Preparation, Infection, Cell Culture, Western Blot, Quantitative RT-PCR

    polyethylenimine transfection  (ATCC)


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    ATCC polyethylenimine transfection
    Polyethylenimine Transfection, supplied by ATCC, 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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    transfection efficiency  (ATCC)


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    ATCC transfection efficiency
    A. U2OS cells were treated with As as indicated and then allowed to recover for the indicated period. Extracts were analysed by western blotting using the indicated antibodies. B. Parental and H6 cells stably expressing GFP-G3BP1 were treated with As (0.2mM) for 1hr before allowed to recover for the indicated periods and fixed. Scale bars, 10μm. C. Quantification of the experiment in B (mean of 3 independent experiments ± SD). D. Similar experiment as in B, using instead parental and H6 U2OS cells and immunostained for endogenous TIA1. E. Quantification of the experiment in D. F. Experiment as described in B, except that cells were prior transfected (48hrs) with empty (pcDNA3) and Nb9 expressing constructs. GFP-G3BP1 staining was used to quantify cells with SGs during the recovery period. G. Parental and H6 U2OS cells stably expressing GFP-G3BP1 pre-treated for 4hrs with 0.5μM NAEi were then treated for 1hr with As (0.2mM), washed with PBS and then incubated in fresh medium containing 0.5μM NAEi for the indicated period before fixation. Graph represents the percentage of cells with SGs (mean ± SD, n=3). H. NEDP1 knockout HEK293 cells (A12) were transfected with the indicated plasmids and 48hrs <t>post-transfection</t> were treated with As (0.2mM) for 1hr before cell extracts were analysed by western blotting with the indicated antibodies. I. NEDP1 KO HEK293 cells (A12) co-transfected with pcDNA3 or NEDP1-WT or NEDP1-Mutant (C163A) and GFP-G3BP1 were treated for 1hr with As (0.2mM) and then allowed to recover for 2.5hrs before fixation and imaging. K. Quantitation of experiment performed in I for percentage of cells with SGs (mean ± SD, n=3).
    Transfection Efficiency, supplied by ATCC, 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 "Targeting the deNEDDylating enzyme NEDP1 to ameliorate ALS phenotypes through Stress Granules dissolution"

    Article Title: Targeting the deNEDDylating enzyme NEDP1 to ameliorate ALS phenotypes through Stress Granules dissolution

    Journal: bioRxiv

    doi: 10.1101/2023.01.06.522988

    A. U2OS cells were treated with As as indicated and then allowed to recover for the indicated period. Extracts were analysed by western blotting using the indicated antibodies. B. Parental and H6 cells stably expressing GFP-G3BP1 were treated with As (0.2mM) for 1hr before allowed to recover for the indicated periods and fixed. Scale bars, 10μm. C. Quantification of the experiment in B (mean of 3 independent experiments ± SD). D. Similar experiment as in B, using instead parental and H6 U2OS cells and immunostained for endogenous TIA1. E. Quantification of the experiment in D. F. Experiment as described in B, except that cells were prior transfected (48hrs) with empty (pcDNA3) and Nb9 expressing constructs. GFP-G3BP1 staining was used to quantify cells with SGs during the recovery period. G. Parental and H6 U2OS cells stably expressing GFP-G3BP1 pre-treated for 4hrs with 0.5μM NAEi were then treated for 1hr with As (0.2mM), washed with PBS and then incubated in fresh medium containing 0.5μM NAEi for the indicated period before fixation. Graph represents the percentage of cells with SGs (mean ± SD, n=3). H. NEDP1 knockout HEK293 cells (A12) were transfected with the indicated plasmids and 48hrs post-transfection were treated with As (0.2mM) for 1hr before cell extracts were analysed by western blotting with the indicated antibodies. I. NEDP1 KO HEK293 cells (A12) co-transfected with pcDNA3 or NEDP1-WT or NEDP1-Mutant (C163A) and GFP-G3BP1 were treated for 1hr with As (0.2mM) and then allowed to recover for 2.5hrs before fixation and imaging. K. Quantitation of experiment performed in I for percentage of cells with SGs (mean ± SD, n=3).
    Figure Legend Snippet: A. U2OS cells were treated with As as indicated and then allowed to recover for the indicated period. Extracts were analysed by western blotting using the indicated antibodies. B. Parental and H6 cells stably expressing GFP-G3BP1 were treated with As (0.2mM) for 1hr before allowed to recover for the indicated periods and fixed. Scale bars, 10μm. C. Quantification of the experiment in B (mean of 3 independent experiments ± SD). D. Similar experiment as in B, using instead parental and H6 U2OS cells and immunostained for endogenous TIA1. E. Quantification of the experiment in D. F. Experiment as described in B, except that cells were prior transfected (48hrs) with empty (pcDNA3) and Nb9 expressing constructs. GFP-G3BP1 staining was used to quantify cells with SGs during the recovery period. G. Parental and H6 U2OS cells stably expressing GFP-G3BP1 pre-treated for 4hrs with 0.5μM NAEi were then treated for 1hr with As (0.2mM), washed with PBS and then incubated in fresh medium containing 0.5μM NAEi for the indicated period before fixation. Graph represents the percentage of cells with SGs (mean ± SD, n=3). H. NEDP1 knockout HEK293 cells (A12) were transfected with the indicated plasmids and 48hrs post-transfection were treated with As (0.2mM) for 1hr before cell extracts were analysed by western blotting with the indicated antibodies. I. NEDP1 KO HEK293 cells (A12) co-transfected with pcDNA3 or NEDP1-WT or NEDP1-Mutant (C163A) and GFP-G3BP1 were treated for 1hr with As (0.2mM) and then allowed to recover for 2.5hrs before fixation and imaging. K. Quantitation of experiment performed in I for percentage of cells with SGs (mean ± SD, n=3).

    Techniques Used: Western Blot, Stable Transfection, Expressing, Transfection, Construct, Staining, Incubation, Knock-Out, Mutagenesis, Imaging, Quantitation Assay

    A. Schematic representation of the used proteomic approach to identify NEDDylation sites controlled by NEDP1 at endogenous levels of wild type NEDD8 expression. Parental and NEDP1 knockout (C8) HCT116 cells were treated with UAEi (0.5 μ M, 5hrs) before extracts were used for trypsin digestion and immunoprecipitations with anti-diglycine antibodies and mass spectrometry analysis. Data were used for label-free quantification. B. Schematic representation of PARP-1 domains including the identified NEDDylated lysine residues upon NEDP1 knockout (this study, ; Lobato et al., 2021). C. Parental and NEDP1 knockout (A12) HEK293 cells stably expressing His6-NEDD8 were treated with As (0.5mM, 1hr) before extracts were used for Nickel-PD. Isolated His6-NEDD8 conjugates and total cell extracts (input) were used for western blot analysis with the indicated antibodies. D. Parental and A12 cells were transfected with His6-PARP-1. 48hrs post-transfection cells were treated with As (0.5mM, 1hr) as indicated and extracts were used for Nickel-PD and western blot analysis as in C. E. HEK293 cells were transfected His6-PARP-1 and Nb9 expression constructs as indicated. 48hrs post-transfection, cells were treated with As (0.5mM, 1hr) and Nickel-PD and western blot analysis was performed as in D. F. A12 HEK293 cells were transfected with the indicated His6-PARP-1 expression constructs and 48hrs later were used for Nickel-PD. Isolated proteins and total cell extracts (input) were used for western blot analysis as indicated. G. Quantitation of the experiment performed in F. Values represent the average of 3 independent experiments ± SD.
    Figure Legend Snippet: A. Schematic representation of the used proteomic approach to identify NEDDylation sites controlled by NEDP1 at endogenous levels of wild type NEDD8 expression. Parental and NEDP1 knockout (C8) HCT116 cells were treated with UAEi (0.5 μ M, 5hrs) before extracts were used for trypsin digestion and immunoprecipitations with anti-diglycine antibodies and mass spectrometry analysis. Data were used for label-free quantification. B. Schematic representation of PARP-1 domains including the identified NEDDylated lysine residues upon NEDP1 knockout (this study, ; Lobato et al., 2021). C. Parental and NEDP1 knockout (A12) HEK293 cells stably expressing His6-NEDD8 were treated with As (0.5mM, 1hr) before extracts were used for Nickel-PD. Isolated His6-NEDD8 conjugates and total cell extracts (input) were used for western blot analysis with the indicated antibodies. D. Parental and A12 cells were transfected with His6-PARP-1. 48hrs post-transfection cells were treated with As (0.5mM, 1hr) as indicated and extracts were used for Nickel-PD and western blot analysis as in C. E. HEK293 cells were transfected His6-PARP-1 and Nb9 expression constructs as indicated. 48hrs post-transfection, cells were treated with As (0.5mM, 1hr) and Nickel-PD and western blot analysis was performed as in D. F. A12 HEK293 cells were transfected with the indicated His6-PARP-1 expression constructs and 48hrs later were used for Nickel-PD. Isolated proteins and total cell extracts (input) were used for western blot analysis as indicated. G. Quantitation of the experiment performed in F. Values represent the average of 3 independent experiments ± SD.

    Techniques Used: Expressing, Knock-Out, Mass Spectrometry, Stable Transfection, Isolation, Western Blot, Transfection, Construct, Quantitation Assay

    A. (Left panels). PARP-1 -/- and PARP-1 -/- / NEDP1 -/- U2OS cells were transfected with the indicated plasmids expressing WT or NEDDylation deficient PARP-1 mutants. 48hrs post-transfection cells were either untreated or treated with As (0.5mM, 1hr) and total cell extracts were used for western blot analysis with the indicated antibodies. (Right panels). Quantitation of the experiment on the left panel. Values represent the mean ± SD from 5 independent experiments of the fraction of PAR production (WT was used as the reference for each condition). B. Parental or H6 U2OS cells treated with As (0.5mM, 1hr) and extracts were used for western blot analysis with the indicated antibodies. C. Experiment was performed as described in A, except that recovery was also employed as indicated. Formation of SGs was monitored by immunostaining for endogenous TIA1. D. In the experiment performed in C, the size of SGs was measured. Values represent the mean of the percentage of cells with the indicated size of granules. E. Quantitation of the experiment performed in C, showing the percentage of cells with SGs during the recovery period. Values represent the mean ± SD from 3 independent experiments.
    Figure Legend Snippet: A. (Left panels). PARP-1 -/- and PARP-1 -/- / NEDP1 -/- U2OS cells were transfected with the indicated plasmids expressing WT or NEDDylation deficient PARP-1 mutants. 48hrs post-transfection cells were either untreated or treated with As (0.5mM, 1hr) and total cell extracts were used for western blot analysis with the indicated antibodies. (Right panels). Quantitation of the experiment on the left panel. Values represent the mean ± SD from 5 independent experiments of the fraction of PAR production (WT was used as the reference for each condition). B. Parental or H6 U2OS cells treated with As (0.5mM, 1hr) and extracts were used for western blot analysis with the indicated antibodies. C. Experiment was performed as described in A, except that recovery was also employed as indicated. Formation of SGs was monitored by immunostaining for endogenous TIA1. D. In the experiment performed in C, the size of SGs was measured. Values represent the mean of the percentage of cells with the indicated size of granules. E. Quantitation of the experiment performed in C, showing the percentage of cells with SGs during the recovery period. Values represent the mean ± SD from 3 independent experiments.

    Techniques Used: Transfection, Expressing, Western Blot, Quantitation Assay, Immunostaining

    A. Parental or A12 (NEDP1 knockout) HEK293 cells were transfected with the indicated GFP-TIA1 constructs (1 μ g) and 48hrs post-transfection cells were stressed with As (0.5mM, 1hr) before recovery (2hrs). SGs formation was monitored by GFP fluorescence. B. Quantitation of the experiment performed in A. Values represent the mean of 3 independent experiments ± SD for the percentage of cells with SGs. C. Experiment performed as in A, except that where indicated the Nb9 was co-transfected. Quantitation was performed as in B. D. Mouse-derived hippocampal neurons were transfected with the indicated GFP-TIA1 and Nb9 constructs, stressed with As (0.2mM, 1hr) before allowed to recover for 2hrs. SGs formation was monitored by GFP fluorescence. E. Scatterplot representing the percentage of co-transfected cells with SGs (mean ± SD). Each dot represents the percentage of co-transfected neurons with SGs on one coverslip (3 coverslips with 20-25 co-transfected neurons per condition). F. Human fibroblasts derived from healthy (WT) donors or patients with sporadic ALS (sALS) were infected with retrovirus expressing NB9 as indicated. Cells were either untreated or treated with As (0.5mM, 1hr) and allowed to recover (1hr). SG formation was monitored by immunostaining for the SG protein eIF4G1. Right panel represents the quantification of the experiment. Values represent the mean ± SD from 3 independent experiments of the percentage of cells with SGs.
    Figure Legend Snippet: A. Parental or A12 (NEDP1 knockout) HEK293 cells were transfected with the indicated GFP-TIA1 constructs (1 μ g) and 48hrs post-transfection cells were stressed with As (0.5mM, 1hr) before recovery (2hrs). SGs formation was monitored by GFP fluorescence. B. Quantitation of the experiment performed in A. Values represent the mean of 3 independent experiments ± SD for the percentage of cells with SGs. C. Experiment performed as in A, except that where indicated the Nb9 was co-transfected. Quantitation was performed as in B. D. Mouse-derived hippocampal neurons were transfected with the indicated GFP-TIA1 and Nb9 constructs, stressed with As (0.2mM, 1hr) before allowed to recover for 2hrs. SGs formation was monitored by GFP fluorescence. E. Scatterplot representing the percentage of co-transfected cells with SGs (mean ± SD). Each dot represents the percentage of co-transfected neurons with SGs on one coverslip (3 coverslips with 20-25 co-transfected neurons per condition). F. Human fibroblasts derived from healthy (WT) donors or patients with sporadic ALS (sALS) were infected with retrovirus expressing NB9 as indicated. Cells were either untreated or treated with As (0.5mM, 1hr) and allowed to recover (1hr). SG formation was monitored by immunostaining for the SG protein eIF4G1. Right panel represents the quantification of the experiment. Values represent the mean ± SD from 3 independent experiments of the percentage of cells with SGs.

    Techniques Used: Knock-Out, Transfection, Construct, Fluorescence, Quantitation Assay, Derivative Assay, Infection, Expressing, Immunostaining

    transfecting hek 293t  (ATCC)


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    ATCC transfecting hek 293t
    a Schematic of relationship between scFv-expressing phage and full-length antibodies. Created with BioRender.com. b Specificity of full-length antibodies grafted from validated scFv-expressing phage. Wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2 or MERS were incubated with converted antibodies. A3_13, which binds to HLA-A3, serves as a negative control for the ELISA. c Measuring neutralization with cPASS assay. Red shaded region represents ≥30% neutralization and is defined as detection of SARS-CoV-2 neutralizing activities. MM42, a non-neutralizing negative control mAb. MM57, a neutralizing positive control mAb. + CTRL, a manufacturer-provided positive control. d Ability of full-length antibodies to inhibit ACE2-spike protein interaction. The converted full-length antibodies were applied to wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2, followed by the addition of recombinant ACE2-His protein. e Ability of full-length antibodies to block pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing SARS-CoV-2 spike protein and ACE2-expressing <t>HEK-293T</t> cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.
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    1) Product Images from "The rapid and highly parallel identification of antibodies with defined biological activities by SLISY"

    Article Title: The rapid and highly parallel identification of antibodies with defined biological activities by SLISY

    Journal: Nature Communications

    doi: 10.1038/s41467-022-35668-6

    a Schematic of relationship between scFv-expressing phage and full-length antibodies. Created with BioRender.com. b Specificity of full-length antibodies grafted from validated scFv-expressing phage. Wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2 or MERS were incubated with converted antibodies. A3_13, which binds to HLA-A3, serves as a negative control for the ELISA. c Measuring neutralization with cPASS assay. Red shaded region represents ≥30% neutralization and is defined as detection of SARS-CoV-2 neutralizing activities. MM42, a non-neutralizing negative control mAb. MM57, a neutralizing positive control mAb. + CTRL, a manufacturer-provided positive control. d Ability of full-length antibodies to inhibit ACE2-spike protein interaction. The converted full-length antibodies were applied to wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2, followed by the addition of recombinant ACE2-His protein. e Ability of full-length antibodies to block pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing SARS-CoV-2 spike protein and ACE2-expressing HEK-293T cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.
    Figure Legend Snippet: a Schematic of relationship between scFv-expressing phage and full-length antibodies. Created with BioRender.com. b Specificity of full-length antibodies grafted from validated scFv-expressing phage. Wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2 or MERS were incubated with converted antibodies. A3_13, which binds to HLA-A3, serves as a negative control for the ELISA. c Measuring neutralization with cPASS assay. Red shaded region represents ≥30% neutralization and is defined as detection of SARS-CoV-2 neutralizing activities. MM42, a non-neutralizing negative control mAb. MM57, a neutralizing positive control mAb. + CTRL, a manufacturer-provided positive control. d Ability of full-length antibodies to inhibit ACE2-spike protein interaction. The converted full-length antibodies were applied to wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2, followed by the addition of recombinant ACE2-His protein. e Ability of full-length antibodies to block pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing SARS-CoV-2 spike protein and ACE2-expressing HEK-293T cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.

    Techniques Used: Expressing, Incubation, Negative Control, Enzyme-linked Immunosorbent Assay, Neutralization, Positive Control, Recombinant, Blocking Assay, Infection, Luciferase, Activity Assay, Standard Deviation

    a Selecting variant binding clones for validation. After applying the enriched pool of scFvs from biopanning with the original SARS-CoV-2 to variant proteins, the original SARS-CoV-2 SBR for each clone was plotted against a variant SBR. Clones were selected for testing based on high SBRs for both the original and variants. Twenty clones were selected from each of the original-variant pairings. b Broad spectrum neutralization by superclone scFvs Beta_10, Gamma_12, and Gamma_19. The full-length scFv phage clones were applied to wells pre-coated with variant SARS-CoV-2 spike proteins, followed by the addition of recombinant ACE2-His protein. Negative control is A3-Clone 13 scFv phage. c Ability of full-length antibodies to block variant pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing variant SARS-CoV-2 spike protein and ACE2-expressing HEK-293T cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.
    Figure Legend Snippet: a Selecting variant binding clones for validation. After applying the enriched pool of scFvs from biopanning with the original SARS-CoV-2 to variant proteins, the original SARS-CoV-2 SBR for each clone was plotted against a variant SBR. Clones were selected for testing based on high SBRs for both the original and variants. Twenty clones were selected from each of the original-variant pairings. b Broad spectrum neutralization by superclone scFvs Beta_10, Gamma_12, and Gamma_19. The full-length scFv phage clones were applied to wells pre-coated with variant SARS-CoV-2 spike proteins, followed by the addition of recombinant ACE2-His protein. Negative control is A3-Clone 13 scFv phage. c Ability of full-length antibodies to block variant pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing variant SARS-CoV-2 spike protein and ACE2-expressing HEK-293T cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.

    Techniques Used: Variant Assay, Binding Assay, Clone Assay, Neutralization, Recombinant, Negative Control, Blocking Assay, Infection, Incubation, Expressing, Luciferase, Activity Assay, Standard Deviation

    lentivirus transfection  (ATCC)


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    ATCC lentivirus transfection
    Lentivirus Transfection, supplied by ATCC, 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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    plasmid transfection  (ATCC)


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    ATCC plasmid transfection
    Plasmid Transfection, supplied by ATCC, 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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    calcium phosphate transfection  (ATCC)


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    ATCC calcium phosphate transfection
    Generation of conditional CHD8 knockout stem cells. ( a ) Strategy to generate a heterozygous conditional knockout (cKO) allele of CHD8 in pluripotent stem cells. The endogenous Exon 4 was flanked with LoxP sites by AAV-mediated homologous recombination. Following correct targeting, the selection cassette was removed by transient <t>transfection</t> with FlpE recombinase to generate the final conditional allele. Infection with Cre recombinase leads to deletion of the floxed allele and generates CHD8 KO cells. Infection with ΔCre (an inactive form of Cre) is used throughout the study for the control condition. ( b ) Generation of homozygous CHD8 cKO cells by introducing a CRISPR transfection-mediated indel mutation into the non-conditional CHD8 allele, which led to a frameshift mutation. Infection of a correctly targeted line with Cre recombinase generates homozygous CHD8 null cells, whereas control infection with ΔCre leaves the engineered mutations unchanged (see also Figs. a–d and a–d) ( c ) Left shows immunofluorescent images of CHD8 staining in conditional heterozygous and homozygous CHD8 KO embryonic stem cells three days after infection with Cre/Δ Cre to detect CHD8 reduction. Right depicts the bright-field images of homozygous CHD8 KO and control neurons, 23 days after in vitro differentiation assay. ( d ) Western blotting of heterozygous, and homozygous neurons (please see also the Fig. d).
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    1) Product Images from "The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1"

    Article Title: The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1

    Journal: Scientific Reports

    doi: 10.1038/s41598-022-23614-x

    Generation of conditional CHD8 knockout stem cells. ( a ) Strategy to generate a heterozygous conditional knockout (cKO) allele of CHD8 in pluripotent stem cells. The endogenous Exon 4 was flanked with LoxP sites by AAV-mediated homologous recombination. Following correct targeting, the selection cassette was removed by transient transfection with FlpE recombinase to generate the final conditional allele. Infection with Cre recombinase leads to deletion of the floxed allele and generates CHD8 KO cells. Infection with ΔCre (an inactive form of Cre) is used throughout the study for the control condition. ( b ) Generation of homozygous CHD8 cKO cells by introducing a CRISPR transfection-mediated indel mutation into the non-conditional CHD8 allele, which led to a frameshift mutation. Infection of a correctly targeted line with Cre recombinase generates homozygous CHD8 null cells, whereas control infection with ΔCre leaves the engineered mutations unchanged (see also Figs. a–d and a–d) ( c ) Left shows immunofluorescent images of CHD8 staining in conditional heterozygous and homozygous CHD8 KO embryonic stem cells three days after infection with Cre/Δ Cre to detect CHD8 reduction. Right depicts the bright-field images of homozygous CHD8 KO and control neurons, 23 days after in vitro differentiation assay. ( d ) Western blotting of heterozygous, and homozygous neurons (please see also the Fig. d).
    Figure Legend Snippet: Generation of conditional CHD8 knockout stem cells. ( a ) Strategy to generate a heterozygous conditional knockout (cKO) allele of CHD8 in pluripotent stem cells. The endogenous Exon 4 was flanked with LoxP sites by AAV-mediated homologous recombination. Following correct targeting, the selection cassette was removed by transient transfection with FlpE recombinase to generate the final conditional allele. Infection with Cre recombinase leads to deletion of the floxed allele and generates CHD8 KO cells. Infection with ΔCre (an inactive form of Cre) is used throughout the study for the control condition. ( b ) Generation of homozygous CHD8 cKO cells by introducing a CRISPR transfection-mediated indel mutation into the non-conditional CHD8 allele, which led to a frameshift mutation. Infection of a correctly targeted line with Cre recombinase generates homozygous CHD8 null cells, whereas control infection with ΔCre leaves the engineered mutations unchanged (see also Figs. a–d and a–d) ( c ) Left shows immunofluorescent images of CHD8 staining in conditional heterozygous and homozygous CHD8 KO embryonic stem cells three days after infection with Cre/Δ Cre to detect CHD8 reduction. Right depicts the bright-field images of homozygous CHD8 KO and control neurons, 23 days after in vitro differentiation assay. ( d ) Western blotting of heterozygous, and homozygous neurons (please see also the Fig. d).

    Techniques Used: Knock-Out, Homologous Recombination, Selection, Transfection, Infection, CRISPR, Mutagenesis, Staining, In Vitro, Differentiation Assay, Western Blot

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    ATCC calcium phosphate co transfection
    ( A ) Illustration of a generic integral membrane protein (representing SYT1 and SYB2) with a luminal HaloTag to allow for selective labeling at the PM. ( B ) Schematic of the HaloTag ligand (HTL) labeling protocol, shown within a nerve terminal, with the non-permeant ligand incubation step to label surface protein. If SYT1 and SYB2 are first delivered to an internal sorting compartment ( i ), rather than the PM ( ii ) prior to SV or SV-intermediate formation, they will not pass through the PM and so will not be labeled by the non-permeant ligand (green). Incubation with permeant ligand labels the remaining tagged protein and the resulting change in fluorescence denotes the efficiency of PM delivery. The signal from labeling with the non-permeant ligand was referred to as F initial , where the unlabeled control coverslips still yielded a small background signal, producing a reproducible non-zero value that allowed us to calculate ratios. The subsequent signal after labeling with permeant ligand was called F final . This labeling step included unbound ligand which, while weak and diffuse, results in a slight increase in the background signal. To counteract this, ROIs were drawn to include only the fluorescence intensity within the synapse. ( C ) Timeline for the <t>transfection</t> and labeling protocols. Briefly, cultured rat hippocampal neurons were transduced with TeTx-LC virus on 5 days in vitro (DIV) and then co-transfected on 9 DIV with HaloTag-SYT1 or SYB2-HaloTag, and SYP-GFP; the GFP construct was included to mark synapses and ‘dilute’ the HaloTag plasmid to achieve lower expression levels. Half of the coverslips were incubated in non-permeant HTL (JF549i) immediately after co-transfection to label any tagged protein that was delivered to the PM. Six days later (15 DIV) neurons were rinsed, imaged, and incubated with permeant ligand (JF549), to label any remaining tagged protein, and imaged again. ( D ) Immunoblot of cells transduced with a virus expressing TeTx-LC, resulting in the cleavage of endogenous SYB2 and the inhibition of SV recycling. We note that the SYB2 fusion protein used in these experiments harbored two point mutations to render it resistant to TeTx-LC (see Methods). Blots were probed for endogenous SYB2, SYT1, and SYP, with a TCE loading control. The normalized (F final /F initial ) change in fluorescence intensity of the SYT1 ( E ) and SYB2 ( F ) fusion proteins upon adding permeant fluorescent ligand to cells grown with or without non-permeant ligand for 6 days; mean values with 95% CI are plotted to the right of each scatter plot. Data were analyzed with unpaired t-tests for both proteins; p-values = <0.0001. Panel ( E ) contains data from 156 synapses cultured in the presence of JF549i, and 136 synapses grown in the absence of this HTL. Data for both groups were from 8 fields of view from 4 different litters. Panel ( F ) contains data from 107 synapses cultured in the presence of JF549i, and 79 synapses grown in the absence of this HTL. Data for both groups were from five fields of view from three different litters. Mean values and descriptive statistics for SYT1 and SYB2 can be found in . Panels ( G, H ) are representative images of SYP-GFP to mark synapses (dashed circles), and the corresponding HaloTag-SYT1 signals under the indicated conditions; in the bottom panels the JF549 ligand was not washed away, resulting in a higher background. For all conditions, identical laser and gain settings were used. Any linear brightness and contrast adjustments were applied to all conditions. ( I, J ) Same as panels ( G ) and ( H ), but for SYB2-HaloTag. Figure 5—source data 1. Descriptive statistics corresponding to . (A) Corresponds to . (B) Corresponds to .
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    ATCC sirna transfections
    ( A ) Illustration of a generic integral membrane protein (representing SYT1 and SYB2) with a luminal HaloTag to allow for selective labeling at the PM. ( B ) Schematic of the HaloTag ligand (HTL) labeling protocol, shown within a nerve terminal, with the non-permeant ligand incubation step to label surface protein. If SYT1 and SYB2 are first delivered to an internal sorting compartment ( i ), rather than the PM ( ii ) prior to SV or SV-intermediate formation, they will not pass through the PM and so will not be labeled by the non-permeant ligand (green). Incubation with permeant ligand labels the remaining tagged protein and the resulting change in fluorescence denotes the efficiency of PM delivery. The signal from labeling with the non-permeant ligand was referred to as F initial , where the unlabeled control coverslips still yielded a small background signal, producing a reproducible non-zero value that allowed us to calculate ratios. The subsequent signal after labeling with permeant ligand was called F final . This labeling step included unbound ligand which, while weak and diffuse, results in a slight increase in the background signal. To counteract this, ROIs were drawn to include only the fluorescence intensity within the synapse. ( C ) Timeline for the <t>transfection</t> and labeling protocols. Briefly, cultured rat hippocampal neurons were transduced with TeTx-LC virus on 5 days in vitro (DIV) and then co-transfected on 9 DIV with HaloTag-SYT1 or SYB2-HaloTag, and SYP-GFP; the GFP construct was included to mark synapses and ‘dilute’ the HaloTag plasmid to achieve lower expression levels. Half of the coverslips were incubated in non-permeant HTL (JF549i) immediately after co-transfection to label any tagged protein that was delivered to the PM. Six days later (15 DIV) neurons were rinsed, imaged, and incubated with permeant ligand (JF549), to label any remaining tagged protein, and imaged again. ( D ) Immunoblot of cells transduced with a virus expressing TeTx-LC, resulting in the cleavage of endogenous SYB2 and the inhibition of SV recycling. We note that the SYB2 fusion protein used in these experiments harbored two point mutations to render it resistant to TeTx-LC (see Methods). Blots were probed for endogenous SYB2, SYT1, and SYP, with a TCE loading control. The normalized (F final /F initial ) change in fluorescence intensity of the SYT1 ( E ) and SYB2 ( F ) fusion proteins upon adding permeant fluorescent ligand to cells grown with or without non-permeant ligand for 6 days; mean values with 95% CI are plotted to the right of each scatter plot. Data were analyzed with unpaired t-tests for both proteins; p-values = <0.0001. Panel ( E ) contains data from 156 synapses cultured in the presence of JF549i, and 136 synapses grown in the absence of this HTL. Data for both groups were from 8 fields of view from 4 different litters. Panel ( F ) contains data from 107 synapses cultured in the presence of JF549i, and 79 synapses grown in the absence of this HTL. Data for both groups were from five fields of view from three different litters. Mean values and descriptive statistics for SYT1 and SYB2 can be found in . Panels ( G, H ) are representative images of SYP-GFP to mark synapses (dashed circles), and the corresponding HaloTag-SYT1 signals under the indicated conditions; in the bottom panels the JF549 ligand was not washed away, resulting in a higher background. For all conditions, identical laser and gain settings were used. Any linear brightness and contrast adjustments were applied to all conditions. ( I, J ) Same as panels ( G ) and ( H ), but for SYB2-HaloTag. Figure 5—source data 1. Descriptive statistics corresponding to . (A) Corresponds to . (B) Corresponds to .
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    ATCC transfex transfection reagent
    ( A ) Illustration of a generic integral membrane protein (representing SYT1 and SYB2) with a luminal HaloTag to allow for selective labeling at the PM. ( B ) Schematic of the HaloTag ligand (HTL) labeling protocol, shown within a nerve terminal, with the non-permeant ligand incubation step to label surface protein. If SYT1 and SYB2 are first delivered to an internal sorting compartment ( i ), rather than the PM ( ii ) prior to SV or SV-intermediate formation, they will not pass through the PM and so will not be labeled by the non-permeant ligand (green). Incubation with permeant ligand labels the remaining tagged protein and the resulting change in fluorescence denotes the efficiency of PM delivery. The signal from labeling with the non-permeant ligand was referred to as F initial , where the unlabeled control coverslips still yielded a small background signal, producing a reproducible non-zero value that allowed us to calculate ratios. The subsequent signal after labeling with permeant ligand was called F final . This labeling step included unbound ligand which, while weak and diffuse, results in a slight increase in the background signal. To counteract this, ROIs were drawn to include only the fluorescence intensity within the synapse. ( C ) Timeline for the <t>transfection</t> and labeling protocols. Briefly, cultured rat hippocampal neurons were transduced with TeTx-LC virus on 5 days in vitro (DIV) and then co-transfected on 9 DIV with HaloTag-SYT1 or SYB2-HaloTag, and SYP-GFP; the GFP construct was included to mark synapses and ‘dilute’ the HaloTag plasmid to achieve lower expression levels. Half of the coverslips were incubated in non-permeant HTL (JF549i) immediately after co-transfection to label any tagged protein that was delivered to the PM. Six days later (15 DIV) neurons were rinsed, imaged, and incubated with permeant ligand (JF549), to label any remaining tagged protein, and imaged again. ( D ) Immunoblot of cells transduced with a virus expressing TeTx-LC, resulting in the cleavage of endogenous SYB2 and the inhibition of SV recycling. We note that the SYB2 fusion protein used in these experiments harbored two point mutations to render it resistant to TeTx-LC (see Methods). Blots were probed for endogenous SYB2, SYT1, and SYP, with a TCE loading control. The normalized (F final /F initial ) change in fluorescence intensity of the SYT1 ( E ) and SYB2 ( F ) fusion proteins upon adding permeant fluorescent ligand to cells grown with or without non-permeant ligand for 6 days; mean values with 95% CI are plotted to the right of each scatter plot. Data were analyzed with unpaired t-tests for both proteins; p-values = <0.0001. Panel ( E ) contains data from 156 synapses cultured in the presence of JF549i, and 136 synapses grown in the absence of this HTL. Data for both groups were from 8 fields of view from 4 different litters. Panel ( F ) contains data from 107 synapses cultured in the presence of JF549i, and 79 synapses grown in the absence of this HTL. Data for both groups were from five fields of view from three different litters. Mean values and descriptive statistics for SYT1 and SYB2 can be found in . Panels ( G, H ) are representative images of SYP-GFP to mark synapses (dashed circles), and the corresponding HaloTag-SYT1 signals under the indicated conditions; in the bottom panels the JF549 ligand was not washed away, resulting in a higher background. For all conditions, identical laser and gain settings were used. Any linear brightness and contrast adjustments were applied to all conditions. ( I, J ) Same as panels ( G ) and ( H ), but for SYB2-HaloTag. Figure 5—source data 1. Descriptive statistics corresponding to . (A) Corresponds to . (B) Corresponds to .
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    ATCC polyfast transfection reagent
    Characterization of du-cGAS functional domains. DEFs cells were transfected with expression plasmids encoding full-length du-cGAS, truncated du-cGAS or empty vector (EV). After 24 h <t>post-transfection,</t> cells were infected with DAdV-B2 at the MOI of 1.0 and harvested at 48 hpi. (A) Cell monolayers were detected the expression of DAdV-B2 hexon protein by IFA. (a) empty plasmid; (b) du-cGAS; (c) du-cGAS (69-423aa) (d) Mab21; (e) Zinc-Ribbon; (f) NTase. (B) The viral titers in the cell culture supernatant were detected by TCID 50 assay. (C) The protein expression of full-length and truncated du-cGAS, and DAdV-B2 hexon was detected by western blotting. (D) The relative mRNA expression of type I IFNs, STING and key ISGs was detected by qRT-PCR. Data represent the mean values ± SD, and statistical significance was analyzed by t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.
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    86
    ATCC polyethylenimine transfection
    Characterization of du-cGAS functional domains. DEFs cells were transfected with expression plasmids encoding full-length du-cGAS, truncated du-cGAS or empty vector (EV). After 24 h <t>post-transfection,</t> cells were infected with DAdV-B2 at the MOI of 1.0 and harvested at 48 hpi. (A) Cell monolayers were detected the expression of DAdV-B2 hexon protein by IFA. (a) empty plasmid; (b) du-cGAS; (c) du-cGAS (69-423aa) (d) Mab21; (e) Zinc-Ribbon; (f) NTase. (B) The viral titers in the cell culture supernatant were detected by TCID 50 assay. (C) The protein expression of full-length and truncated du-cGAS, and DAdV-B2 hexon was detected by western blotting. (D) The relative mRNA expression of type I IFNs, STING and key ISGs was detected by qRT-PCR. Data represent the mean values ± SD, and statistical significance was analyzed by t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.
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    ATCC transfection efficiency
    A. U2OS cells were treated with As as indicated and then allowed to recover for the indicated period. Extracts were analysed by western blotting using the indicated antibodies. B. Parental and H6 cells stably expressing GFP-G3BP1 were treated with As (0.2mM) for 1hr before allowed to recover for the indicated periods and fixed. Scale bars, 10μm. C. Quantification of the experiment in B (mean of 3 independent experiments ± SD). D. Similar experiment as in B, using instead parental and H6 U2OS cells and immunostained for endogenous TIA1. E. Quantification of the experiment in D. F. Experiment as described in B, except that cells were prior transfected (48hrs) with empty (pcDNA3) and Nb9 expressing constructs. GFP-G3BP1 staining was used to quantify cells with SGs during the recovery period. G. Parental and H6 U2OS cells stably expressing GFP-G3BP1 pre-treated for 4hrs with 0.5μM NAEi were then treated for 1hr with As (0.2mM), washed with PBS and then incubated in fresh medium containing 0.5μM NAEi for the indicated period before fixation. Graph represents the percentage of cells with SGs (mean ± SD, n=3). H. NEDP1 knockout HEK293 cells (A12) were transfected with the indicated plasmids and 48hrs <t>post-transfection</t> were treated with As (0.2mM) for 1hr before cell extracts were analysed by western blotting with the indicated antibodies. I. NEDP1 KO HEK293 cells (A12) co-transfected with pcDNA3 or NEDP1-WT or NEDP1-Mutant (C163A) and GFP-G3BP1 were treated for 1hr with As (0.2mM) and then allowed to recover for 2.5hrs before fixation and imaging. K. Quantitation of experiment performed in I for percentage of cells with SGs (mean ± SD, n=3).
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    86
    ATCC transfecting hek 293t
    a Schematic of relationship between scFv-expressing phage and full-length antibodies. Created with BioRender.com. b Specificity of full-length antibodies grafted from validated scFv-expressing phage. Wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2 or MERS were incubated with converted antibodies. A3_13, which binds to HLA-A3, serves as a negative control for the ELISA. c Measuring neutralization with cPASS assay. Red shaded region represents ≥30% neutralization and is defined as detection of SARS-CoV-2 neutralizing activities. MM42, a non-neutralizing negative control mAb. MM57, a neutralizing positive control mAb. + CTRL, a manufacturer-provided positive control. d Ability of full-length antibodies to inhibit ACE2-spike protein interaction. The converted full-length antibodies were applied to wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2, followed by the addition of recombinant ACE2-His protein. e Ability of full-length antibodies to block pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing SARS-CoV-2 spike protein and ACE2-expressing <t>HEK-293T</t> cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.
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    ATCC lentivirus transfection
    a Schematic of relationship between scFv-expressing phage and full-length antibodies. Created with BioRender.com. b Specificity of full-length antibodies grafted from validated scFv-expressing phage. Wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2 or MERS were incubated with converted antibodies. A3_13, which binds to HLA-A3, serves as a negative control for the ELISA. c Measuring neutralization with cPASS assay. Red shaded region represents ≥30% neutralization and is defined as detection of SARS-CoV-2 neutralizing activities. MM42, a non-neutralizing negative control mAb. MM57, a neutralizing positive control mAb. + CTRL, a manufacturer-provided positive control. d Ability of full-length antibodies to inhibit ACE2-spike protein interaction. The converted full-length antibodies were applied to wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2, followed by the addition of recombinant ACE2-His protein. e Ability of full-length antibodies to block pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing SARS-CoV-2 spike protein and ACE2-expressing <t>HEK-293T</t> cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.
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    ATCC plasmid transfection
    a Schematic of relationship between scFv-expressing phage and full-length antibodies. Created with BioRender.com. b Specificity of full-length antibodies grafted from validated scFv-expressing phage. Wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2 or MERS were incubated with converted antibodies. A3_13, which binds to HLA-A3, serves as a negative control for the ELISA. c Measuring neutralization with cPASS assay. Red shaded region represents ≥30% neutralization and is defined as detection of SARS-CoV-2 neutralizing activities. MM42, a non-neutralizing negative control mAb. MM57, a neutralizing positive control mAb. + CTRL, a manufacturer-provided positive control. d Ability of full-length antibodies to inhibit ACE2-spike protein interaction. The converted full-length antibodies were applied to wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2, followed by the addition of recombinant ACE2-His protein. e Ability of full-length antibodies to block pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing SARS-CoV-2 spike protein and ACE2-expressing <t>HEK-293T</t> cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.
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    ATCC calcium phosphate transfection
    Generation of conditional CHD8 knockout stem cells. ( a ) Strategy to generate a heterozygous conditional knockout (cKO) allele of CHD8 in pluripotent stem cells. The endogenous Exon 4 was flanked with LoxP sites by AAV-mediated homologous recombination. Following correct targeting, the selection cassette was removed by transient <t>transfection</t> with FlpE recombinase to generate the final conditional allele. Infection with Cre recombinase leads to deletion of the floxed allele and generates CHD8 KO cells. Infection with ΔCre (an inactive form of Cre) is used throughout the study for the control condition. ( b ) Generation of homozygous CHD8 cKO cells by introducing a CRISPR transfection-mediated indel mutation into the non-conditional CHD8 allele, which led to a frameshift mutation. Infection of a correctly targeted line with Cre recombinase generates homozygous CHD8 null cells, whereas control infection with ΔCre leaves the engineered mutations unchanged (see also Figs. a–d and a–d) ( c ) Left shows immunofluorescent images of CHD8 staining in conditional heterozygous and homozygous CHD8 KO embryonic stem cells three days after infection with Cre/Δ Cre to detect CHD8 reduction. Right depicts the bright-field images of homozygous CHD8 KO and control neurons, 23 days after in vitro differentiation assay. ( d ) Western blotting of heterozygous, and homozygous neurons (please see also the Fig. d).
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    Image Search Results


    ( A ) Illustration of a generic integral membrane protein (representing SYT1 and SYB2) with a luminal HaloTag to allow for selective labeling at the PM. ( B ) Schematic of the HaloTag ligand (HTL) labeling protocol, shown within a nerve terminal, with the non-permeant ligand incubation step to label surface protein. If SYT1 and SYB2 are first delivered to an internal sorting compartment ( i ), rather than the PM ( ii ) prior to SV or SV-intermediate formation, they will not pass through the PM and so will not be labeled by the non-permeant ligand (green). Incubation with permeant ligand labels the remaining tagged protein and the resulting change in fluorescence denotes the efficiency of PM delivery. The signal from labeling with the non-permeant ligand was referred to as F initial , where the unlabeled control coverslips still yielded a small background signal, producing a reproducible non-zero value that allowed us to calculate ratios. The subsequent signal after labeling with permeant ligand was called F final . This labeling step included unbound ligand which, while weak and diffuse, results in a slight increase in the background signal. To counteract this, ROIs were drawn to include only the fluorescence intensity within the synapse. ( C ) Timeline for the transfection and labeling protocols. Briefly, cultured rat hippocampal neurons were transduced with TeTx-LC virus on 5 days in vitro (DIV) and then co-transfected on 9 DIV with HaloTag-SYT1 or SYB2-HaloTag, and SYP-GFP; the GFP construct was included to mark synapses and ‘dilute’ the HaloTag plasmid to achieve lower expression levels. Half of the coverslips were incubated in non-permeant HTL (JF549i) immediately after co-transfection to label any tagged protein that was delivered to the PM. Six days later (15 DIV) neurons were rinsed, imaged, and incubated with permeant ligand (JF549), to label any remaining tagged protein, and imaged again. ( D ) Immunoblot of cells transduced with a virus expressing TeTx-LC, resulting in the cleavage of endogenous SYB2 and the inhibition of SV recycling. We note that the SYB2 fusion protein used in these experiments harbored two point mutations to render it resistant to TeTx-LC (see Methods). Blots were probed for endogenous SYB2, SYT1, and SYP, with a TCE loading control. The normalized (F final /F initial ) change in fluorescence intensity of the SYT1 ( E ) and SYB2 ( F ) fusion proteins upon adding permeant fluorescent ligand to cells grown with or without non-permeant ligand for 6 days; mean values with 95% CI are plotted to the right of each scatter plot. Data were analyzed with unpaired t-tests for both proteins; p-values = <0.0001. Panel ( E ) contains data from 156 synapses cultured in the presence of JF549i, and 136 synapses grown in the absence of this HTL. Data for both groups were from 8 fields of view from 4 different litters. Panel ( F ) contains data from 107 synapses cultured in the presence of JF549i, and 79 synapses grown in the absence of this HTL. Data for both groups were from five fields of view from three different litters. Mean values and descriptive statistics for SYT1 and SYB2 can be found in . Panels ( G, H ) are representative images of SYP-GFP to mark synapses (dashed circles), and the corresponding HaloTag-SYT1 signals under the indicated conditions; in the bottom panels the JF549 ligand was not washed away, resulting in a higher background. For all conditions, identical laser and gain settings were used. Any linear brightness and contrast adjustments were applied to all conditions. ( I, J ) Same as panels ( G ) and ( H ), but for SYB2-HaloTag. Figure 5—source data 1. Descriptive statistics corresponding to . (A) Corresponds to . (B) Corresponds to .

    Journal: eLife

    Article Title: Synaptic vesicle proteins are selectively delivered to axons in mammalian neurons

    doi: 10.7554/eLife.82568

    Figure Lengend Snippet: ( A ) Illustration of a generic integral membrane protein (representing SYT1 and SYB2) with a luminal HaloTag to allow for selective labeling at the PM. ( B ) Schematic of the HaloTag ligand (HTL) labeling protocol, shown within a nerve terminal, with the non-permeant ligand incubation step to label surface protein. If SYT1 and SYB2 are first delivered to an internal sorting compartment ( i ), rather than the PM ( ii ) prior to SV or SV-intermediate formation, they will not pass through the PM and so will not be labeled by the non-permeant ligand (green). Incubation with permeant ligand labels the remaining tagged protein and the resulting change in fluorescence denotes the efficiency of PM delivery. The signal from labeling with the non-permeant ligand was referred to as F initial , where the unlabeled control coverslips still yielded a small background signal, producing a reproducible non-zero value that allowed us to calculate ratios. The subsequent signal after labeling with permeant ligand was called F final . This labeling step included unbound ligand which, while weak and diffuse, results in a slight increase in the background signal. To counteract this, ROIs were drawn to include only the fluorescence intensity within the synapse. ( C ) Timeline for the transfection and labeling protocols. Briefly, cultured rat hippocampal neurons were transduced with TeTx-LC virus on 5 days in vitro (DIV) and then co-transfected on 9 DIV with HaloTag-SYT1 or SYB2-HaloTag, and SYP-GFP; the GFP construct was included to mark synapses and ‘dilute’ the HaloTag plasmid to achieve lower expression levels. Half of the coverslips were incubated in non-permeant HTL (JF549i) immediately after co-transfection to label any tagged protein that was delivered to the PM. Six days later (15 DIV) neurons were rinsed, imaged, and incubated with permeant ligand (JF549), to label any remaining tagged protein, and imaged again. ( D ) Immunoblot of cells transduced with a virus expressing TeTx-LC, resulting in the cleavage of endogenous SYB2 and the inhibition of SV recycling. We note that the SYB2 fusion protein used in these experiments harbored two point mutations to render it resistant to TeTx-LC (see Methods). Blots were probed for endogenous SYB2, SYT1, and SYP, with a TCE loading control. The normalized (F final /F initial ) change in fluorescence intensity of the SYT1 ( E ) and SYB2 ( F ) fusion proteins upon adding permeant fluorescent ligand to cells grown with or without non-permeant ligand for 6 days; mean values with 95% CI are plotted to the right of each scatter plot. Data were analyzed with unpaired t-tests for both proteins; p-values = <0.0001. Panel ( E ) contains data from 156 synapses cultured in the presence of JF549i, and 136 synapses grown in the absence of this HTL. Data for both groups were from 8 fields of view from 4 different litters. Panel ( F ) contains data from 107 synapses cultured in the presence of JF549i, and 79 synapses grown in the absence of this HTL. Data for both groups were from five fields of view from three different litters. Mean values and descriptive statistics for SYT1 and SYB2 can be found in . Panels ( G, H ) are representative images of SYP-GFP to mark synapses (dashed circles), and the corresponding HaloTag-SYT1 signals under the indicated conditions; in the bottom panels the JF549 ligand was not washed away, resulting in a higher background. For all conditions, identical laser and gain settings were used. Any linear brightness and contrast adjustments were applied to all conditions. ( I, J ) Same as panels ( G ) and ( H ), but for SYB2-HaloTag. Figure 5—source data 1. Descriptive statistics corresponding to . (A) Corresponds to . (B) Corresponds to .

    Article Snippet: Lentiviral particles were generated via calcium phosphate co-transfection of HEK293T cells (ATCC, CRL-3216; RRID: CVCL_0063 ) at 30–40% confluency with the pFUGW transfer plasmid and the packaging plasmids, pCD/NL-BH*DDD and pLTR-G. Plasmids pCD/NL-BH*DDD (Addgene plasmid #17531; http://n2t.net/addgene :17531; RRID: Addgene_17531 ) ( ) and pLTR-G (Addgene plasmid #17532; http://n2t.net/addgene :17532; RRID: Addgene_17532 ) ( ) were gifts from J Reiser (Bethesda, MD).

    Techniques: Labeling, Incubation, Fluorescence, Transfection, Cell Culture, Transduction, In Vitro, Construct, Plasmid Preparation, Expressing, Cotransfection, Western Blot, Inhibition

    Characterization of du-cGAS functional domains. DEFs cells were transfected with expression plasmids encoding full-length du-cGAS, truncated du-cGAS or empty vector (EV). After 24 h post-transfection, cells were infected with DAdV-B2 at the MOI of 1.0 and harvested at 48 hpi. (A) Cell monolayers were detected the expression of DAdV-B2 hexon protein by IFA. (a) empty plasmid; (b) du-cGAS; (c) du-cGAS (69-423aa) (d) Mab21; (e) Zinc-Ribbon; (f) NTase. (B) The viral titers in the cell culture supernatant were detected by TCID 50 assay. (C) The protein expression of full-length and truncated du-cGAS, and DAdV-B2 hexon was detected by western blotting. (D) The relative mRNA expression of type I IFNs, STING and key ISGs was detected by qRT-PCR. Data represent the mean values ± SD, and statistical significance was analyzed by t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.

    Journal: Frontiers in Immunology

    Article Title: Duck cGAS inhibits DNA and RNA virus replication by activating IFNs and antiviral ISGs

    doi: 10.3389/fimmu.2023.1101335

    Figure Lengend Snippet: Characterization of du-cGAS functional domains. DEFs cells were transfected with expression plasmids encoding full-length du-cGAS, truncated du-cGAS or empty vector (EV). After 24 h post-transfection, cells were infected with DAdV-B2 at the MOI of 1.0 and harvested at 48 hpi. (A) Cell monolayers were detected the expression of DAdV-B2 hexon protein by IFA. (a) empty plasmid; (b) du-cGAS; (c) du-cGAS (69-423aa) (d) Mab21; (e) Zinc-Ribbon; (f) NTase. (B) The viral titers in the cell culture supernatant were detected by TCID 50 assay. (C) The protein expression of full-length and truncated du-cGAS, and DAdV-B2 hexon was detected by western blotting. (D) The relative mRNA expression of type I IFNs, STING and key ISGs was detected by qRT-PCR. Data represent the mean values ± SD, and statistical significance was analyzed by t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.

    Article Snippet: To generate the cGAS-knockout DEF cells, we transfected Du-cGAS-px459 into ATCC DEF using PolyFast Transfection Reagent (MCG, US), according to the manufacturer’s instructions.

    Techniques: Functional Assay, Transfection, Expressing, Plasmid Preparation, Infection, Cell Culture, Western Blot, Quantitative RT-PCR

    A. U2OS cells were treated with As as indicated and then allowed to recover for the indicated period. Extracts were analysed by western blotting using the indicated antibodies. B. Parental and H6 cells stably expressing GFP-G3BP1 were treated with As (0.2mM) for 1hr before allowed to recover for the indicated periods and fixed. Scale bars, 10μm. C. Quantification of the experiment in B (mean of 3 independent experiments ± SD). D. Similar experiment as in B, using instead parental and H6 U2OS cells and immunostained for endogenous TIA1. E. Quantification of the experiment in D. F. Experiment as described in B, except that cells were prior transfected (48hrs) with empty (pcDNA3) and Nb9 expressing constructs. GFP-G3BP1 staining was used to quantify cells with SGs during the recovery period. G. Parental and H6 U2OS cells stably expressing GFP-G3BP1 pre-treated for 4hrs with 0.5μM NAEi were then treated for 1hr with As (0.2mM), washed with PBS and then incubated in fresh medium containing 0.5μM NAEi for the indicated period before fixation. Graph represents the percentage of cells with SGs (mean ± SD, n=3). H. NEDP1 knockout HEK293 cells (A12) were transfected with the indicated plasmids and 48hrs post-transfection were treated with As (0.2mM) for 1hr before cell extracts were analysed by western blotting with the indicated antibodies. I. NEDP1 KO HEK293 cells (A12) co-transfected with pcDNA3 or NEDP1-WT or NEDP1-Mutant (C163A) and GFP-G3BP1 were treated for 1hr with As (0.2mM) and then allowed to recover for 2.5hrs before fixation and imaging. K. Quantitation of experiment performed in I for percentage of cells with SGs (mean ± SD, n=3).

    Journal: bioRxiv

    Article Title: Targeting the deNEDDylating enzyme NEDP1 to ameliorate ALS phenotypes through Stress Granules dissolution

    doi: 10.1101/2023.01.06.522988

    Figure Lengend Snippet: A. U2OS cells were treated with As as indicated and then allowed to recover for the indicated period. Extracts were analysed by western blotting using the indicated antibodies. B. Parental and H6 cells stably expressing GFP-G3BP1 were treated with As (0.2mM) for 1hr before allowed to recover for the indicated periods and fixed. Scale bars, 10μm. C. Quantification of the experiment in B (mean of 3 independent experiments ± SD). D. Similar experiment as in B, using instead parental and H6 U2OS cells and immunostained for endogenous TIA1. E. Quantification of the experiment in D. F. Experiment as described in B, except that cells were prior transfected (48hrs) with empty (pcDNA3) and Nb9 expressing constructs. GFP-G3BP1 staining was used to quantify cells with SGs during the recovery period. G. Parental and H6 U2OS cells stably expressing GFP-G3BP1 pre-treated for 4hrs with 0.5μM NAEi were then treated for 1hr with As (0.2mM), washed with PBS and then incubated in fresh medium containing 0.5μM NAEi for the indicated period before fixation. Graph represents the percentage of cells with SGs (mean ± SD, n=3). H. NEDP1 knockout HEK293 cells (A12) were transfected with the indicated plasmids and 48hrs post-transfection were treated with As (0.2mM) for 1hr before cell extracts were analysed by western blotting with the indicated antibodies. I. NEDP1 KO HEK293 cells (A12) co-transfected with pcDNA3 or NEDP1-WT or NEDP1-Mutant (C163A) and GFP-G3BP1 were treated for 1hr with As (0.2mM) and then allowed to recover for 2.5hrs before fixation and imaging. K. Quantitation of experiment performed in I for percentage of cells with SGs (mean ± SD, n=3).

    Article Snippet: Cell lines, U2OS (Female) preferred system for live imaging, HCT116 (Female) preferred system for diGly proteomics based on previous studies (44), HEK293 (Male), preferred system for transient over-expression studies due to high transfection efficiency were originally obtained from the ATCC bioresource.

    Techniques: Western Blot, Stable Transfection, Expressing, Transfection, Construct, Staining, Incubation, Knock-Out, Mutagenesis, Imaging, Quantitation Assay

    A. Schematic representation of the used proteomic approach to identify NEDDylation sites controlled by NEDP1 at endogenous levels of wild type NEDD8 expression. Parental and NEDP1 knockout (C8) HCT116 cells were treated with UAEi (0.5 μ M, 5hrs) before extracts were used for trypsin digestion and immunoprecipitations with anti-diglycine antibodies and mass spectrometry analysis. Data were used for label-free quantification. B. Schematic representation of PARP-1 domains including the identified NEDDylated lysine residues upon NEDP1 knockout (this study, ; Lobato et al., 2021). C. Parental and NEDP1 knockout (A12) HEK293 cells stably expressing His6-NEDD8 were treated with As (0.5mM, 1hr) before extracts were used for Nickel-PD. Isolated His6-NEDD8 conjugates and total cell extracts (input) were used for western blot analysis with the indicated antibodies. D. Parental and A12 cells were transfected with His6-PARP-1. 48hrs post-transfection cells were treated with As (0.5mM, 1hr) as indicated and extracts were used for Nickel-PD and western blot analysis as in C. E. HEK293 cells were transfected His6-PARP-1 and Nb9 expression constructs as indicated. 48hrs post-transfection, cells were treated with As (0.5mM, 1hr) and Nickel-PD and western blot analysis was performed as in D. F. A12 HEK293 cells were transfected with the indicated His6-PARP-1 expression constructs and 48hrs later were used for Nickel-PD. Isolated proteins and total cell extracts (input) were used for western blot analysis as indicated. G. Quantitation of the experiment performed in F. Values represent the average of 3 independent experiments ± SD.

    Journal: bioRxiv

    Article Title: Targeting the deNEDDylating enzyme NEDP1 to ameliorate ALS phenotypes through Stress Granules dissolution

    doi: 10.1101/2023.01.06.522988

    Figure Lengend Snippet: A. Schematic representation of the used proteomic approach to identify NEDDylation sites controlled by NEDP1 at endogenous levels of wild type NEDD8 expression. Parental and NEDP1 knockout (C8) HCT116 cells were treated with UAEi (0.5 μ M, 5hrs) before extracts were used for trypsin digestion and immunoprecipitations with anti-diglycine antibodies and mass spectrometry analysis. Data were used for label-free quantification. B. Schematic representation of PARP-1 domains including the identified NEDDylated lysine residues upon NEDP1 knockout (this study, ; Lobato et al., 2021). C. Parental and NEDP1 knockout (A12) HEK293 cells stably expressing His6-NEDD8 were treated with As (0.5mM, 1hr) before extracts were used for Nickel-PD. Isolated His6-NEDD8 conjugates and total cell extracts (input) were used for western blot analysis with the indicated antibodies. D. Parental and A12 cells were transfected with His6-PARP-1. 48hrs post-transfection cells were treated with As (0.5mM, 1hr) as indicated and extracts were used for Nickel-PD and western blot analysis as in C. E. HEK293 cells were transfected His6-PARP-1 and Nb9 expression constructs as indicated. 48hrs post-transfection, cells were treated with As (0.5mM, 1hr) and Nickel-PD and western blot analysis was performed as in D. F. A12 HEK293 cells were transfected with the indicated His6-PARP-1 expression constructs and 48hrs later were used for Nickel-PD. Isolated proteins and total cell extracts (input) were used for western blot analysis as indicated. G. Quantitation of the experiment performed in F. Values represent the average of 3 independent experiments ± SD.

    Article Snippet: Cell lines, U2OS (Female) preferred system for live imaging, HCT116 (Female) preferred system for diGly proteomics based on previous studies (44), HEK293 (Male), preferred system for transient over-expression studies due to high transfection efficiency were originally obtained from the ATCC bioresource.

    Techniques: Expressing, Knock-Out, Mass Spectrometry, Stable Transfection, Isolation, Western Blot, Transfection, Construct, Quantitation Assay

    A. (Left panels). PARP-1 -/- and PARP-1 -/- / NEDP1 -/- U2OS cells were transfected with the indicated plasmids expressing WT or NEDDylation deficient PARP-1 mutants. 48hrs post-transfection cells were either untreated or treated with As (0.5mM, 1hr) and total cell extracts were used for western blot analysis with the indicated antibodies. (Right panels). Quantitation of the experiment on the left panel. Values represent the mean ± SD from 5 independent experiments of the fraction of PAR production (WT was used as the reference for each condition). B. Parental or H6 U2OS cells treated with As (0.5mM, 1hr) and extracts were used for western blot analysis with the indicated antibodies. C. Experiment was performed as described in A, except that recovery was also employed as indicated. Formation of SGs was monitored by immunostaining for endogenous TIA1. D. In the experiment performed in C, the size of SGs was measured. Values represent the mean of the percentage of cells with the indicated size of granules. E. Quantitation of the experiment performed in C, showing the percentage of cells with SGs during the recovery period. Values represent the mean ± SD from 3 independent experiments.

    Journal: bioRxiv

    Article Title: Targeting the deNEDDylating enzyme NEDP1 to ameliorate ALS phenotypes through Stress Granules dissolution

    doi: 10.1101/2023.01.06.522988

    Figure Lengend Snippet: A. (Left panels). PARP-1 -/- and PARP-1 -/- / NEDP1 -/- U2OS cells were transfected with the indicated plasmids expressing WT or NEDDylation deficient PARP-1 mutants. 48hrs post-transfection cells were either untreated or treated with As (0.5mM, 1hr) and total cell extracts were used for western blot analysis with the indicated antibodies. (Right panels). Quantitation of the experiment on the left panel. Values represent the mean ± SD from 5 independent experiments of the fraction of PAR production (WT was used as the reference for each condition). B. Parental or H6 U2OS cells treated with As (0.5mM, 1hr) and extracts were used for western blot analysis with the indicated antibodies. C. Experiment was performed as described in A, except that recovery was also employed as indicated. Formation of SGs was monitored by immunostaining for endogenous TIA1. D. In the experiment performed in C, the size of SGs was measured. Values represent the mean of the percentage of cells with the indicated size of granules. E. Quantitation of the experiment performed in C, showing the percentage of cells with SGs during the recovery period. Values represent the mean ± SD from 3 independent experiments.

    Article Snippet: Cell lines, U2OS (Female) preferred system for live imaging, HCT116 (Female) preferred system for diGly proteomics based on previous studies (44), HEK293 (Male), preferred system for transient over-expression studies due to high transfection efficiency were originally obtained from the ATCC bioresource.

    Techniques: Transfection, Expressing, Western Blot, Quantitation Assay, Immunostaining

    A. Parental or A12 (NEDP1 knockout) HEK293 cells were transfected with the indicated GFP-TIA1 constructs (1 μ g) and 48hrs post-transfection cells were stressed with As (0.5mM, 1hr) before recovery (2hrs). SGs formation was monitored by GFP fluorescence. B. Quantitation of the experiment performed in A. Values represent the mean of 3 independent experiments ± SD for the percentage of cells with SGs. C. Experiment performed as in A, except that where indicated the Nb9 was co-transfected. Quantitation was performed as in B. D. Mouse-derived hippocampal neurons were transfected with the indicated GFP-TIA1 and Nb9 constructs, stressed with As (0.2mM, 1hr) before allowed to recover for 2hrs. SGs formation was monitored by GFP fluorescence. E. Scatterplot representing the percentage of co-transfected cells with SGs (mean ± SD). Each dot represents the percentage of co-transfected neurons with SGs on one coverslip (3 coverslips with 20-25 co-transfected neurons per condition). F. Human fibroblasts derived from healthy (WT) donors or patients with sporadic ALS (sALS) were infected with retrovirus expressing NB9 as indicated. Cells were either untreated or treated with As (0.5mM, 1hr) and allowed to recover (1hr). SG formation was monitored by immunostaining for the SG protein eIF4G1. Right panel represents the quantification of the experiment. Values represent the mean ± SD from 3 independent experiments of the percentage of cells with SGs.

    Journal: bioRxiv

    Article Title: Targeting the deNEDDylating enzyme NEDP1 to ameliorate ALS phenotypes through Stress Granules dissolution

    doi: 10.1101/2023.01.06.522988

    Figure Lengend Snippet: A. Parental or A12 (NEDP1 knockout) HEK293 cells were transfected with the indicated GFP-TIA1 constructs (1 μ g) and 48hrs post-transfection cells were stressed with As (0.5mM, 1hr) before recovery (2hrs). SGs formation was monitored by GFP fluorescence. B. Quantitation of the experiment performed in A. Values represent the mean of 3 independent experiments ± SD for the percentage of cells with SGs. C. Experiment performed as in A, except that where indicated the Nb9 was co-transfected. Quantitation was performed as in B. D. Mouse-derived hippocampal neurons were transfected with the indicated GFP-TIA1 and Nb9 constructs, stressed with As (0.2mM, 1hr) before allowed to recover for 2hrs. SGs formation was monitored by GFP fluorescence. E. Scatterplot representing the percentage of co-transfected cells with SGs (mean ± SD). Each dot represents the percentage of co-transfected neurons with SGs on one coverslip (3 coverslips with 20-25 co-transfected neurons per condition). F. Human fibroblasts derived from healthy (WT) donors or patients with sporadic ALS (sALS) were infected with retrovirus expressing NB9 as indicated. Cells were either untreated or treated with As (0.5mM, 1hr) and allowed to recover (1hr). SG formation was monitored by immunostaining for the SG protein eIF4G1. Right panel represents the quantification of the experiment. Values represent the mean ± SD from 3 independent experiments of the percentage of cells with SGs.

    Article Snippet: Cell lines, U2OS (Female) preferred system for live imaging, HCT116 (Female) preferred system for diGly proteomics based on previous studies (44), HEK293 (Male), preferred system for transient over-expression studies due to high transfection efficiency were originally obtained from the ATCC bioresource.

    Techniques: Knock-Out, Transfection, Construct, Fluorescence, Quantitation Assay, Derivative Assay, Infection, Expressing, Immunostaining

    a Schematic of relationship between scFv-expressing phage and full-length antibodies. Created with BioRender.com. b Specificity of full-length antibodies grafted from validated scFv-expressing phage. Wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2 or MERS were incubated with converted antibodies. A3_13, which binds to HLA-A3, serves as a negative control for the ELISA. c Measuring neutralization with cPASS assay. Red shaded region represents ≥30% neutralization and is defined as detection of SARS-CoV-2 neutralizing activities. MM42, a non-neutralizing negative control mAb. MM57, a neutralizing positive control mAb. + CTRL, a manufacturer-provided positive control. d Ability of full-length antibodies to inhibit ACE2-spike protein interaction. The converted full-length antibodies were applied to wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2, followed by the addition of recombinant ACE2-His protein. e Ability of full-length antibodies to block pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing SARS-CoV-2 spike protein and ACE2-expressing HEK-293T cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: The rapid and highly parallel identification of antibodies with defined biological activities by SLISY

    doi: 10.1038/s41467-022-35668-6

    Figure Lengend Snippet: a Schematic of relationship between scFv-expressing phage and full-length antibodies. Created with BioRender.com. b Specificity of full-length antibodies grafted from validated scFv-expressing phage. Wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2 or MERS were incubated with converted antibodies. A3_13, which binds to HLA-A3, serves as a negative control for the ELISA. c Measuring neutralization with cPASS assay. Red shaded region represents ≥30% neutralization and is defined as detection of SARS-CoV-2 neutralizing activities. MM42, a non-neutralizing negative control mAb. MM57, a neutralizing positive control mAb. + CTRL, a manufacturer-provided positive control. d Ability of full-length antibodies to inhibit ACE2-spike protein interaction. The converted full-length antibodies were applied to wells pre-coated with RBD, S1 or FL spike proteins from SARS-CoV-2, followed by the addition of recombinant ACE2-His protein. e Ability of full-length antibodies to block pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing SARS-CoV-2 spike protein and ACE2-expressing HEK-293T cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.

    Article Snippet: SARS-CoV-2 S-protein pseudotyped replication incompetent lentiviral particles were produced by first transfecting HEK-293T (ATCC, CRL-3216) with GeneJuice transfection reagent (Millipore-Sigma) and SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentiviral Kit (Spike-Pseudotyped Lentiviral Kit, NR-52948; BEI Resources Repository, Manassas, VA).

    Techniques: Expressing, Incubation, Negative Control, Enzyme-linked Immunosorbent Assay, Neutralization, Positive Control, Recombinant, Blocking Assay, Infection, Luciferase, Activity Assay, Standard Deviation

    a Selecting variant binding clones for validation. After applying the enriched pool of scFvs from biopanning with the original SARS-CoV-2 to variant proteins, the original SARS-CoV-2 SBR for each clone was plotted against a variant SBR. Clones were selected for testing based on high SBRs for both the original and variants. Twenty clones were selected from each of the original-variant pairings. b Broad spectrum neutralization by superclone scFvs Beta_10, Gamma_12, and Gamma_19. The full-length scFv phage clones were applied to wells pre-coated with variant SARS-CoV-2 spike proteins, followed by the addition of recombinant ACE2-His protein. Negative control is A3-Clone 13 scFv phage. c Ability of full-length antibodies to block variant pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing variant SARS-CoV-2 spike protein and ACE2-expressing HEK-293T cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: The rapid and highly parallel identification of antibodies with defined biological activities by SLISY

    doi: 10.1038/s41467-022-35668-6

    Figure Lengend Snippet: a Selecting variant binding clones for validation. After applying the enriched pool of scFvs from biopanning with the original SARS-CoV-2 to variant proteins, the original SARS-CoV-2 SBR for each clone was plotted against a variant SBR. Clones were selected for testing based on high SBRs for both the original and variants. Twenty clones were selected from each of the original-variant pairings. b Broad spectrum neutralization by superclone scFvs Beta_10, Gamma_12, and Gamma_19. The full-length scFv phage clones were applied to wells pre-coated with variant SARS-CoV-2 spike proteins, followed by the addition of recombinant ACE2-His protein. Negative control is A3-Clone 13 scFv phage. c Ability of full-length antibodies to block variant pseudovirus infectivity. Converted full-length antibodies were incubated with pseudovirus expressing variant SARS-CoV-2 spike protein and ACE2-expressing HEK-293T cells for 48 h. Infectivity was measured by luciferase activity. Error bars represent standard deviation of means ( n = 3). Source data are provided as a Source Data file.

    Article Snippet: SARS-CoV-2 S-protein pseudotyped replication incompetent lentiviral particles were produced by first transfecting HEK-293T (ATCC, CRL-3216) with GeneJuice transfection reagent (Millipore-Sigma) and SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike-Pseudotyped Lentiviral Kit (Spike-Pseudotyped Lentiviral Kit, NR-52948; BEI Resources Repository, Manassas, VA).

    Techniques: Variant Assay, Binding Assay, Clone Assay, Neutralization, Recombinant, Negative Control, Blocking Assay, Infection, Incubation, Expressing, Luciferase, Activity Assay, Standard Deviation

    Generation of conditional CHD8 knockout stem cells. ( a ) Strategy to generate a heterozygous conditional knockout (cKO) allele of CHD8 in pluripotent stem cells. The endogenous Exon 4 was flanked with LoxP sites by AAV-mediated homologous recombination. Following correct targeting, the selection cassette was removed by transient transfection with FlpE recombinase to generate the final conditional allele. Infection with Cre recombinase leads to deletion of the floxed allele and generates CHD8 KO cells. Infection with ΔCre (an inactive form of Cre) is used throughout the study for the control condition. ( b ) Generation of homozygous CHD8 cKO cells by introducing a CRISPR transfection-mediated indel mutation into the non-conditional CHD8 allele, which led to a frameshift mutation. Infection of a correctly targeted line with Cre recombinase generates homozygous CHD8 null cells, whereas control infection with ΔCre leaves the engineered mutations unchanged (see also Figs. a–d and a–d) ( c ) Left shows immunofluorescent images of CHD8 staining in conditional heterozygous and homozygous CHD8 KO embryonic stem cells three days after infection with Cre/Δ Cre to detect CHD8 reduction. Right depicts the bright-field images of homozygous CHD8 KO and control neurons, 23 days after in vitro differentiation assay. ( d ) Western blotting of heterozygous, and homozygous neurons (please see also the Fig. d).

    Journal: Scientific Reports

    Article Title: The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1

    doi: 10.1038/s41598-022-23614-x

    Figure Lengend Snippet: Generation of conditional CHD8 knockout stem cells. ( a ) Strategy to generate a heterozygous conditional knockout (cKO) allele of CHD8 in pluripotent stem cells. The endogenous Exon 4 was flanked with LoxP sites by AAV-mediated homologous recombination. Following correct targeting, the selection cassette was removed by transient transfection with FlpE recombinase to generate the final conditional allele. Infection with Cre recombinase leads to deletion of the floxed allele and generates CHD8 KO cells. Infection with ΔCre (an inactive form of Cre) is used throughout the study for the control condition. ( b ) Generation of homozygous CHD8 cKO cells by introducing a CRISPR transfection-mediated indel mutation into the non-conditional CHD8 allele, which led to a frameshift mutation. Infection of a correctly targeted line with Cre recombinase generates homozygous CHD8 null cells, whereas control infection with ΔCre leaves the engineered mutations unchanged (see also Figs. a–d and a–d) ( c ) Left shows immunofluorescent images of CHD8 staining in conditional heterozygous and homozygous CHD8 KO embryonic stem cells three days after infection with Cre/Δ Cre to detect CHD8 reduction. Right depicts the bright-field images of homozygous CHD8 KO and control neurons, 23 days after in vitro differentiation assay. ( d ) Western blotting of heterozygous, and homozygous neurons (please see also the Fig. d).

    Article Snippet: To produce rAAV, we co-transfected three plasmids: 25 µg of pAAV , 25 µg of a helper plasmid (pAd5), and 20 µg of the capsid (AAV-DJ), into one T75 flask with 80% confluent HEK293T cells (ATCC) by calcium phosphate transfection method , .

    Techniques: Knock-Out, Homologous Recombination, Selection, Transfection, Infection, CRISPR, Mutagenesis, Staining, In Vitro, Differentiation Assay, Western Blot