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  • 88
    ATUM codon optimized open reading frame
    Codon Optimized Open Reading Frame, supplied by ATUM, used in various techniques. Bioz Stars score: 88/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/codon optimized open reading frame/product/ATUM
    Average 88 stars, based on 22 article reviews
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    codon optimized open reading frame - by Bioz Stars, 2020-08
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    86
    ATUM codon optimized mif open reading frame
    Codon Optimized Mif Open Reading Frame, supplied by ATUM, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/codon optimized mif open reading frame/product/ATUM
    Average 86 stars, based on 9 article reviews
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    codon optimized mif open reading frame - by Bioz Stars, 2020-08
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    85
    Bio Basic Canada human codon optimized open reading frames
    Human Codon Optimized Open Reading Frames, supplied by Bio Basic Canada, used in various techniques. Bioz Stars score: 85/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 14 article reviews
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    human codon optimized open reading frames - by Bioz Stars, 2020-08
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    91
    Thermo Fisher codon optimized open reading frames
    Codon Optimized Open Reading Frames, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 34 article reviews
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    codon optimized open reading frames - by Bioz Stars, 2020-08
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    91
    GenScript codon optimized open reading frame
    Codon Optimized Open Reading Frame, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 1 article reviews
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    codon optimized open reading frame - by Bioz Stars, 2020-08
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    93
    Addgene inc codon optimized lbcas12a open reading frame
    Improved multiplex editing, gene activation, and base editing with enAsCas12a. ( a - c ) Comparison of the multiplex modification efficiencies of AsCas12a, enAsCas12a, and <t>LbCas12a,</t> when programmed with TTTV PAM targeted crRNA arrays encoding 3 separate crRNAs expressed either from a polymerase III promoter (U6, panels a and b ) or a polymerase II promoter (CAG, panel c ). The activities at three separate loci were assessed by T7E1 assay using the same genomic DNA samples; mean, s.e.m., and individual data points shown for n = 3. ( d ) Assessment of editing efficiencies with AsCas12a, enAsCas12a, and LbCas12a when using pooled crRNA plasmids or multiplex crRNA arrays expressing two crRNAs targeted to nearby (~100 bp) genomic loci. Activities assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n = 4. ( e-g ), Activation of endogenous human genes NPY1R , HBB , and AR with dCas12a-VPR(1.1) fusions in HEK293 cells using pools of three crRNAs targeted to canonical PAM sites ( panel e ) and non-canonical PAM sites ( panels f and g ). Activities assessed by RT-qPCR and fold-changes in RNA were normalized to HPRT1 levels; mean, s.e.m., and individual data points shown for three independent experiments (mean of three technical triplicate qPCR values); VPR, synthetic VP64-p65-Rta activation domain 26 . ( h ) Cytosine to thymine (C-to-T) conversion efficiencies directed by dCas12a base-editor (BE) constructs across eight different target sites, assessed by targeted deep sequencing. The mean percent C-to-T editing of three independent experiments was examined within a −5 to +25 window; all Cs in this window are highlighted in green for each target site; the position of the C within the target site is indicated below the heat map. ( i ) C-to-T editing efficiency within the 20 nt target site spacer sequence with enAsBEs and LbBEs across all eight target sites.
    Codon Optimized Lbcas12a Open Reading Frame, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GenScript codon optimized fab30 open reading frame
    Ligand-dependent modulation of core interaction in Apo β 2 V 2 R phos <t>–βarr1-Fab30</t> complex. ( a ) A schematic representation showing that Apo β 2 V 2 R phos can potentially sample active like conformations and, therefore, might engage core interaction to some extent. Incubation with an inverse agonist is likely to ablate this basal level of core interaction yielding a ‘tail only' complex while incubation with an agonist stabilizes the core interaction and results in a ‘fully engaged' complex. ( b ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as assessed by ELISA approach. Incubation of this pre-formed complex with inverse agonist or agonist does not alter the physical assembly of the complex. ( c ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as measured by coimmunoprecipitation. Similar to ELISA approach, incubation of pre-formed complex with inverse agonist or agonist does not alter the complex assembly. This experiment was repeated three times with identical results and a representative image is shown. ( d ) Incubation of pre-formed Apo β 2 V 2 R phos complex with inverse agonist (carazolol) results in an increase in bimane fluorescence suggesting a loss of core binding, yet presumably stabilization of a ‘tail engaged' complex. On the other hand, incubation of this complex with agonist (BI-167107) results in a further decrease in bimane fluorescence suggesting the engagement of receptor core and, therefore, stabilization of a ‘fully engaged' complex. ( e ) Bimane fluorescence at emission λ max as measured in d is presented as a bar graph. ( f ) Incubation of pre-formed Apo β 2 V 2 R phos complex with a panel of ligands results in different extent of bimane fluorescence quenching, which directly correlates to the ligand efficacy. ( g ) Quantification of decrease in bimane fluorescence at emission λ max as measured in f is presented as a bar graph. Data in d and f represent mean of three independent experiments. Data presented in b , e and g represent mean±s.e.m. of three independent experiments and analysed using one-way ANOVA with Bonferroni post-test (* P
    Codon Optimized Fab30 Open Reading Frame, supplied by GenScript, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa codon optimized egfp open reading frame orf
    Ligand-dependent modulation of core interaction in Apo β 2 V 2 R phos <t>–βarr1-Fab30</t> complex. ( a ) A schematic representation showing that Apo β 2 V 2 R phos can potentially sample active like conformations and, therefore, might engage core interaction to some extent. Incubation with an inverse agonist is likely to ablate this basal level of core interaction yielding a ‘tail only' complex while incubation with an agonist stabilizes the core interaction and results in a ‘fully engaged' complex. ( b ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as assessed by ELISA approach. Incubation of this pre-formed complex with inverse agonist or agonist does not alter the physical assembly of the complex. ( c ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as measured by coimmunoprecipitation. Similar to ELISA approach, incubation of pre-formed complex with inverse agonist or agonist does not alter the complex assembly. This experiment was repeated three times with identical results and a representative image is shown. ( d ) Incubation of pre-formed Apo β 2 V 2 R phos complex with inverse agonist (carazolol) results in an increase in bimane fluorescence suggesting a loss of core binding, yet presumably stabilization of a ‘tail engaged' complex. On the other hand, incubation of this complex with agonist (BI-167107) results in a further decrease in bimane fluorescence suggesting the engagement of receptor core and, therefore, stabilization of a ‘fully engaged' complex. ( e ) Bimane fluorescence at emission λ max as measured in d is presented as a bar graph. ( f ) Incubation of pre-formed Apo β 2 V 2 R phos complex with a panel of ligands results in different extent of bimane fluorescence quenching, which directly correlates to the ligand efficacy. ( g ) Quantification of decrease in bimane fluorescence at emission λ max as measured in f is presented as a bar graph. Data in d and f represent mean of three independent experiments. Data presented in b , e and g represent mean±s.e.m. of three independent experiments and analysed using one-way ANOVA with Bonferroni post-test (* P
    Codon Optimized Egfp Open Reading Frame Orf, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 3 article reviews
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    86
    Thermo Fisher codon optimized xmrv gag open reading frame
    Ligand-dependent modulation of core interaction in Apo β 2 V 2 R phos <t>–βarr1-Fab30</t> complex. ( a ) A schematic representation showing that Apo β 2 V 2 R phos can potentially sample active like conformations and, therefore, might engage core interaction to some extent. Incubation with an inverse agonist is likely to ablate this basal level of core interaction yielding a ‘tail only' complex while incubation with an agonist stabilizes the core interaction and results in a ‘fully engaged' complex. ( b ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as assessed by ELISA approach. Incubation of this pre-formed complex with inverse agonist or agonist does not alter the physical assembly of the complex. ( c ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as measured by coimmunoprecipitation. Similar to ELISA approach, incubation of pre-formed complex with inverse agonist or agonist does not alter the complex assembly. This experiment was repeated three times with identical results and a representative image is shown. ( d ) Incubation of pre-formed Apo β 2 V 2 R phos complex with inverse agonist (carazolol) results in an increase in bimane fluorescence suggesting a loss of core binding, yet presumably stabilization of a ‘tail engaged' complex. On the other hand, incubation of this complex with agonist (BI-167107) results in a further decrease in bimane fluorescence suggesting the engagement of receptor core and, therefore, stabilization of a ‘fully engaged' complex. ( e ) Bimane fluorescence at emission λ max as measured in d is presented as a bar graph. ( f ) Incubation of pre-formed Apo β 2 V 2 R phos complex with a panel of ligands results in different extent of bimane fluorescence quenching, which directly correlates to the ligand efficacy. ( g ) Quantification of decrease in bimane fluorescence at emission λ max as measured in f is presented as a bar graph. Data in d and f represent mean of three independent experiments. Data presented in b , e and g represent mean±s.e.m. of three independent experiments and analysed using one-way ANOVA with Bonferroni post-test (* P
    Codon Optimized Xmrv Gag Open Reading Frame, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher codon optimized open reading frame encoding naga 100083g23
    Ligand-dependent modulation of core interaction in Apo β 2 V 2 R phos <t>–βarr1-Fab30</t> complex. ( a ) A schematic representation showing that Apo β 2 V 2 R phos can potentially sample active like conformations and, therefore, might engage core interaction to some extent. Incubation with an inverse agonist is likely to ablate this basal level of core interaction yielding a ‘tail only' complex while incubation with an agonist stabilizes the core interaction and results in a ‘fully engaged' complex. ( b ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as assessed by ELISA approach. Incubation of this pre-formed complex with inverse agonist or agonist does not alter the physical assembly of the complex. ( c ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as measured by coimmunoprecipitation. Similar to ELISA approach, incubation of pre-formed complex with inverse agonist or agonist does not alter the complex assembly. This experiment was repeated three times with identical results and a representative image is shown. ( d ) Incubation of pre-formed Apo β 2 V 2 R phos complex with inverse agonist (carazolol) results in an increase in bimane fluorescence suggesting a loss of core binding, yet presumably stabilization of a ‘tail engaged' complex. On the other hand, incubation of this complex with agonist (BI-167107) results in a further decrease in bimane fluorescence suggesting the engagement of receptor core and, therefore, stabilization of a ‘fully engaged' complex. ( e ) Bimane fluorescence at emission λ max as measured in d is presented as a bar graph. ( f ) Incubation of pre-formed Apo β 2 V 2 R phos complex with a panel of ligands results in different extent of bimane fluorescence quenching, which directly correlates to the ligand efficacy. ( g ) Quantification of decrease in bimane fluorescence at emission λ max as measured in f is presented as a bar graph. Data in d and f represent mean of three independent experiments. Data presented in b , e and g represent mean±s.e.m. of three independent experiments and analysed using one-way ANOVA with Bonferroni post-test (* P
    Codon Optimized Open Reading Frame Encoding Naga 100083g23, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/codon optimized open reading frame encoding naga 100083g23/product/Thermo Fisher
    Average 90 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    codon optimized open reading frame encoding naga 100083g23 - by Bioz Stars, 2020-08
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    90
    Thermo Fisher codon optimized gapa gene open reading frame
    Ligand-dependent modulation of core interaction in Apo β 2 V 2 R phos <t>–βarr1-Fab30</t> complex. ( a ) A schematic representation showing that Apo β 2 V 2 R phos can potentially sample active like conformations and, therefore, might engage core interaction to some extent. Incubation with an inverse agonist is likely to ablate this basal level of core interaction yielding a ‘tail only' complex while incubation with an agonist stabilizes the core interaction and results in a ‘fully engaged' complex. ( b ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as assessed by ELISA approach. Incubation of this pre-formed complex with inverse agonist or agonist does not alter the physical assembly of the complex. ( c ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as measured by coimmunoprecipitation. Similar to ELISA approach, incubation of pre-formed complex with inverse agonist or agonist does not alter the complex assembly. This experiment was repeated three times with identical results and a representative image is shown. ( d ) Incubation of pre-formed Apo β 2 V 2 R phos complex with inverse agonist (carazolol) results in an increase in bimane fluorescence suggesting a loss of core binding, yet presumably stabilization of a ‘tail engaged' complex. On the other hand, incubation of this complex with agonist (BI-167107) results in a further decrease in bimane fluorescence suggesting the engagement of receptor core and, therefore, stabilization of a ‘fully engaged' complex. ( e ) Bimane fluorescence at emission λ max as measured in d is presented as a bar graph. ( f ) Incubation of pre-formed Apo β 2 V 2 R phos complex with a panel of ligands results in different extent of bimane fluorescence quenching, which directly correlates to the ligand efficacy. ( g ) Quantification of decrease in bimane fluorescence at emission λ max as measured in f is presented as a bar graph. Data in d and f represent mean of three independent experiments. Data presented in b , e and g represent mean±s.e.m. of three independent experiments and analysed using one-way ANOVA with Bonferroni post-test (* P
    Codon Optimized Gapa Gene Open Reading Frame, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 6 article reviews
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    84
    GenScript recombinant lsm2 8 codon optimized open reading frames
    Structures of S. pombe <t>Lsm2-8</t> bound to pentauridylate with a 2’,3’ OH or 2’,3’ cyclic phosphate. A) Overview of Lsm2-8 bound to “unprocessed” U6 snRNA 3’ terminus. B) Overview of Lsm2-8 bound to “mature” U6 snRNA 3’ terminus. C) Detail of Lsm2-8 interface with “unprocessed” RNA. The 3’ terminal uridine is disordered and thus not visible in the final electron density maps. D) In contrast, the mature U6 3’ end, with a 2’,3′ cyclic phosphate, shows electron density for the terminal nucleotide. E) The Sm-like pocket in Lsm3 binds RNA as observed previously in other Lsm2-8 complexes from S. cerevisiae . F) In contrast, the 3′ uridine cyclic phosphate has a unique binding mechanism relative to the other four uridines, including a stacking interaction with the C-terminal histidine of Lsm8.
    Recombinant Lsm2 8 Codon Optimized Open Reading Frames, supplied by GenScript, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GenScript codon optimized orf
    Structures of S. pombe <t>Lsm2-8</t> bound to pentauridylate with a 2’,3’ OH or 2’,3’ cyclic phosphate. A) Overview of Lsm2-8 bound to “unprocessed” U6 snRNA 3’ terminus. B) Overview of Lsm2-8 bound to “mature” U6 snRNA 3’ terminus. C) Detail of Lsm2-8 interface with “unprocessed” RNA. The 3’ terminal uridine is disordered and thus not visible in the final electron density maps. D) In contrast, the mature U6 3’ end, with a 2’,3′ cyclic phosphate, shows electron density for the terminal nucleotide. E) The Sm-like pocket in Lsm3 binds RNA as observed previously in other Lsm2-8 complexes from S. cerevisiae . F) In contrast, the 3′ uridine cyclic phosphate has a unique binding mechanism relative to the other four uridines, including a stacking interaction with the C-terminal histidine of Lsm8.
    Codon Optimized Orf, supplied by GenScript, used in various techniques. Bioz Stars score: 90/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    codon optimized orf - by Bioz Stars, 2020-08
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    91
    Thermo Fisher human codon optimized orf ul89
    Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, <t>UL89,</t> or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 <t>ORF</t> or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.
    Human Codon Optimized Orf Ul89, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Mr. Gene GmbH codon optimized hant4 orf
    Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, <t>UL89,</t> or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 <t>ORF</t> or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.
    Codon Optimized Hant4 Orf, supplied by Mr. Gene GmbH, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/codon optimized hant4 orf/product/Mr. Gene GmbH
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    88
    Thermo Fisher codon optimized h5 coh5 orf
    Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, <t>UL89,</t> or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 <t>ORF</t> or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.
    Codon Optimized H5 Coh5 Orf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    codon optimized h5 coh5 orf - by Bioz Stars, 2020-08
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    93
    Thermo Fisher codon optimized hazv l orf
    Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, <t>UL89,</t> or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 <t>ORF</t> or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.
    Codon Optimized Hazv L Orf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, <t>UL89,</t> or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 <t>ORF</t> or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.
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    Thermo Fisher codon optimized i3 coi3 orf
    Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, <t>UL89,</t> or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 <t>ORF</t> or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.
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    GenScript codon optimized e2 orf
    Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, <t>UL89,</t> or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 <t>ORF</t> or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.
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    Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, <t>UL89,</t> or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 <t>ORF</t> or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.
    Codon Optimization, supplied by GenScript, used in various techniques. Bioz Stars score: 94/100, based on 1464 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc codon optimized mcpyv vp1 orf
    Density gradient centrifugation of SV40 and MCVSyn particles. Optiprep™ gradient centrifugation was performed with cell lysates from CV1 (A) and H1299 (B) cells 4d after transfection with viral DNA. 15×250 µl fractions were collected (fraction 1 represents the fraction with the highest density and fraction 15 represents the lowest density fraction). (A) Left panel: Real time PCR of micrococcal nuclease treated fractions was performed using SV40 <t>VP1</t> primer sequences. 20 µl of each gradient fraction was loaded on a 10% SDS-page followed immunoblotting using anti-VP1 serum. Right panel: Negative EM staining of SV40 particles identified in fraction 9. (B) Left panel: Real time PCR results of H1299 MCVSyn gradient fractions after micrococcal nuclease treatment using <t>MCPyV</t> VP1-specific primers. Right panel: Negative EM staining of particles identified in fractions 10 and 6.
    Codon Optimized Mcpyv Vp1 Orf, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenScript e coli codon optimized variant
    Density gradient centrifugation of SV40 and MCVSyn particles. Optiprep™ gradient centrifugation was performed with cell lysates from CV1 (A) and H1299 (B) cells 4d after transfection with viral DNA. 15×250 µl fractions were collected (fraction 1 represents the fraction with the highest density and fraction 15 represents the lowest density fraction). (A) Left panel: Real time PCR of micrococcal nuclease treated fractions was performed using SV40 <t>VP1</t> primer sequences. 20 µl of each gradient fraction was loaded on a 10% SDS-page followed immunoblotting using anti-VP1 serum. Right panel: Negative EM staining of SV40 particles identified in fraction 9. (B) Left panel: Real time PCR results of H1299 MCVSyn gradient fractions after micrococcal nuclease treatment using <t>MCPyV</t> VP1-specific primers. Right panel: Negative EM staining of particles identified in fractions 10 and 6.
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    Thermo Fisher codon optimized orf2 expression plasmid
    Retrotransposition driven by full-length L1 elements containing mutations in putative phosphorylation sites within <t>ORF2.</t> a Alu retrotransposition: Full-length L1 elements containing the indicated putative phosphorylation mutations within the ORF2 sequence were used to drive Alu retrotransposition in HeLa cells, as previously described [ 3 ]. L1 is the functional element and L1 EN- is a non-functional element containing a mutation in the ORF2 endonuclease domain (D205A). Control indicates cells transfected with an empty vector and the Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by Neo R colonies (Y-axis). b L1 retrotransposition: Full-length L1 elements containing the indicated putative phosphorylation mutations within the ORF2 sequence were used in an L1 retrotransposition assay in HeLa cells, as previously described [ 5 , 56 ]. L1 is the functional element and control indicates cells transfected with an empty plasmid. The graph depicts the relative number of L1 retrotransposition events as represented by Neo R colonies (Y-axis)
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    Image Search Results


    Improved multiplex editing, gene activation, and base editing with enAsCas12a. ( a - c ) Comparison of the multiplex modification efficiencies of AsCas12a, enAsCas12a, and LbCas12a, when programmed with TTTV PAM targeted crRNA arrays encoding 3 separate crRNAs expressed either from a polymerase III promoter (U6, panels a and b ) or a polymerase II promoter (CAG, panel c ). The activities at three separate loci were assessed by T7E1 assay using the same genomic DNA samples; mean, s.e.m., and individual data points shown for n = 3. ( d ) Assessment of editing efficiencies with AsCas12a, enAsCas12a, and LbCas12a when using pooled crRNA plasmids or multiplex crRNA arrays expressing two crRNAs targeted to nearby (~100 bp) genomic loci. Activities assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n = 4. ( e-g ), Activation of endogenous human genes NPY1R , HBB , and AR with dCas12a-VPR(1.1) fusions in HEK293 cells using pools of three crRNAs targeted to canonical PAM sites ( panel e ) and non-canonical PAM sites ( panels f and g ). Activities assessed by RT-qPCR and fold-changes in RNA were normalized to HPRT1 levels; mean, s.e.m., and individual data points shown for three independent experiments (mean of three technical triplicate qPCR values); VPR, synthetic VP64-p65-Rta activation domain 26 . ( h ) Cytosine to thymine (C-to-T) conversion efficiencies directed by dCas12a base-editor (BE) constructs across eight different target sites, assessed by targeted deep sequencing. The mean percent C-to-T editing of three independent experiments was examined within a −5 to +25 window; all Cs in this window are highlighted in green for each target site; the position of the C within the target site is indicated below the heat map. ( i ) C-to-T editing efficiency within the 20 nt target site spacer sequence with enAsBEs and LbBEs across all eight target sites.

    Journal: Nature biotechnology

    Article Title: Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing

    doi: 10.1038/s41587-018-0011-0

    Figure Lengend Snippet: Improved multiplex editing, gene activation, and base editing with enAsCas12a. ( a - c ) Comparison of the multiplex modification efficiencies of AsCas12a, enAsCas12a, and LbCas12a, when programmed with TTTV PAM targeted crRNA arrays encoding 3 separate crRNAs expressed either from a polymerase III promoter (U6, panels a and b ) or a polymerase II promoter (CAG, panel c ). The activities at three separate loci were assessed by T7E1 assay using the same genomic DNA samples; mean, s.e.m., and individual data points shown for n = 3. ( d ) Assessment of editing efficiencies with AsCas12a, enAsCas12a, and LbCas12a when using pooled crRNA plasmids or multiplex crRNA arrays expressing two crRNAs targeted to nearby (~100 bp) genomic loci. Activities assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n = 4. ( e-g ), Activation of endogenous human genes NPY1R , HBB , and AR with dCas12a-VPR(1.1) fusions in HEK293 cells using pools of three crRNAs targeted to canonical PAM sites ( panel e ) and non-canonical PAM sites ( panels f and g ). Activities assessed by RT-qPCR and fold-changes in RNA were normalized to HPRT1 levels; mean, s.e.m., and individual data points shown for three independent experiments (mean of three technical triplicate qPCR values); VPR, synthetic VP64-p65-Rta activation domain 26 . ( h ) Cytosine to thymine (C-to-T) conversion efficiencies directed by dCas12a base-editor (BE) constructs across eight different target sites, assessed by targeted deep sequencing. The mean percent C-to-T editing of three independent experiments was examined within a −5 to +25 window; all Cs in this window are highlighted in green for each target site; the position of the C within the target site is indicated below the heat map. ( i ) C-to-T editing efficiency within the 20 nt target site spacer sequence with enAsBEs and LbBEs across all eight target sites.

    Article Snippet: Protein expression plasmids were generated by cloning the human codon-optimized open reading frame of AsCas12a and the bacterial codon-optimized LbCas12a open reading frame (from Addgene plasmid 79008; a gift from Jin Soo Kim) into the NcoI and FseI sites of pET28b-Cas9 (Addgene plasmid 47327; a gift from Alex Schier) to generate BPK3541 and RTW645, respectively.

    Techniques: Multiplex Assay, Activation Assay, Modification, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Construct, Sequencing

    Ligand-dependent modulation of core interaction in Apo β 2 V 2 R phos –βarr1-Fab30 complex. ( a ) A schematic representation showing that Apo β 2 V 2 R phos can potentially sample active like conformations and, therefore, might engage core interaction to some extent. Incubation with an inverse agonist is likely to ablate this basal level of core interaction yielding a ‘tail only' complex while incubation with an agonist stabilizes the core interaction and results in a ‘fully engaged' complex. ( b ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as assessed by ELISA approach. Incubation of this pre-formed complex with inverse agonist or agonist does not alter the physical assembly of the complex. ( c ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as measured by coimmunoprecipitation. Similar to ELISA approach, incubation of pre-formed complex with inverse agonist or agonist does not alter the complex assembly. This experiment was repeated three times with identical results and a representative image is shown. ( d ) Incubation of pre-formed Apo β 2 V 2 R phos complex with inverse agonist (carazolol) results in an increase in bimane fluorescence suggesting a loss of core binding, yet presumably stabilization of a ‘tail engaged' complex. On the other hand, incubation of this complex with agonist (BI-167107) results in a further decrease in bimane fluorescence suggesting the engagement of receptor core and, therefore, stabilization of a ‘fully engaged' complex. ( e ) Bimane fluorescence at emission λ max as measured in d is presented as a bar graph. ( f ) Incubation of pre-formed Apo β 2 V 2 R phos complex with a panel of ligands results in different extent of bimane fluorescence quenching, which directly correlates to the ligand efficacy. ( g ) Quantification of decrease in bimane fluorescence at emission λ max as measured in f is presented as a bar graph. Data in d and f represent mean of three independent experiments. Data presented in b , e and g represent mean±s.e.m. of three independent experiments and analysed using one-way ANOVA with Bonferroni post-test (* P

    Journal: Nature Communications

    Article Title: Functional competence of a partially engaged GPCR–β-arrestin complex

    doi: 10.1038/ncomms13416

    Figure Lengend Snippet: Ligand-dependent modulation of core interaction in Apo β 2 V 2 R phos –βarr1-Fab30 complex. ( a ) A schematic representation showing that Apo β 2 V 2 R phos can potentially sample active like conformations and, therefore, might engage core interaction to some extent. Incubation with an inverse agonist is likely to ablate this basal level of core interaction yielding a ‘tail only' complex while incubation with an agonist stabilizes the core interaction and results in a ‘fully engaged' complex. ( b ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as assessed by ELISA approach. Incubation of this pre-formed complex with inverse agonist or agonist does not alter the physical assembly of the complex. ( c ) In-vitro assembly of Apo β 2 V 2 R phos complex with βarr1 in presence of Fab30 as measured by coimmunoprecipitation. Similar to ELISA approach, incubation of pre-formed complex with inverse agonist or agonist does not alter the complex assembly. This experiment was repeated three times with identical results and a representative image is shown. ( d ) Incubation of pre-formed Apo β 2 V 2 R phos complex with inverse agonist (carazolol) results in an increase in bimane fluorescence suggesting a loss of core binding, yet presumably stabilization of a ‘tail engaged' complex. On the other hand, incubation of this complex with agonist (BI-167107) results in a further decrease in bimane fluorescence suggesting the engagement of receptor core and, therefore, stabilization of a ‘fully engaged' complex. ( e ) Bimane fluorescence at emission λ max as measured in d is presented as a bar graph. ( f ) Incubation of pre-formed Apo β 2 V 2 R phos complex with a panel of ligands results in different extent of bimane fluorescence quenching, which directly correlates to the ligand efficacy. ( g ) Quantification of decrease in bimane fluorescence at emission λ max as measured in f is presented as a bar graph. Data in d and f represent mean of three independent experiments. Data presented in b , e and g represent mean±s.e.m. of three independent experiments and analysed using one-way ANOVA with Bonferroni post-test (* P

    Article Snippet: Codon optimized Fab30 open reading frame was synthesized (Genscript) based on published crystal structure (PDB ID: 4JQI) (ref. ), expressed and purified in M55244 strain of E. coli (purchased from American Type Culture Collection) .

    Techniques: Incubation, In Vitro, Enzyme-linked Immunosorbent Assay, Fluorescence, Binding Assay

    A βarr biased ligand of β 2 AR does not promote core interaction with βarr1. ( a ) Carvedilol is a high-affinity βarr biased ligand of β 2 AR and it selectively promotes βarr binding and ERK activation in the absence of any detectable G protein coupling. ( b ) Carvedilol bound and phosphorylated β 2 V 2 R (referred to as Bias β 2 V 2 R phos generated through incubation of Apo β 2 V 2 R phos with tenfold molar excess of carvedilol) exhibits a robust interaction with βarr1 in the presence of Fab30 as assessed by ELISA. Purified Fab30 was immobilized and then incubated with βarr1 and either Bias β 2 V 2 R phos or Act β 2 V 2 R phos . Formation of complex was detected using anti-FLAG M2 antibody. ( c ) Formation of βarr1 complex with Bias β 2 V 2 R phos in the presence of Fab30 as assessed by coimmunoprecipitation. The experiment was repeated three times with identical results and a representative image is shown. Quantification of the data is shown as bar graph. ( d ) Interaction of Bias β 2 AR phos +βarr1+ScFv30 and Act β 2 V 2 R phos +βarr1+ScFv30 complexes with inactive and ( e ) active ERK2. Purified ERK2 was immobilized followed by incubation with pre-formed complexes and detection using HRP-coupled anti-FLAG M2 antibody. ( f ) Interaction of Bias β 2 V 2 R phos with βarr1 does not lead to a detectable decrease in bimane fluorescence suggesting the lack of core interaction. Apo β 2 V 2 R phos was first incubated with tenfold molar excess of carvedilol or BI-167107 to obtain Bias β 2 V 2 R phos and Act β 2 V 2 R phos , respectively. Subsequently, these receptor preparations were incubated with bimane labelled βarr1 and Fab 30 to form a complex followed by fluorescence scanning in the wavelength range indicated on the graph. The data represent an average of three independent experiments. Data presented in b , d and e represent mean±s.e.m. of at least three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (** P

    Journal: Nature Communications

    Article Title: Functional competence of a partially engaged GPCR–β-arrestin complex

    doi: 10.1038/ncomms13416

    Figure Lengend Snippet: A βarr biased ligand of β 2 AR does not promote core interaction with βarr1. ( a ) Carvedilol is a high-affinity βarr biased ligand of β 2 AR and it selectively promotes βarr binding and ERK activation in the absence of any detectable G protein coupling. ( b ) Carvedilol bound and phosphorylated β 2 V 2 R (referred to as Bias β 2 V 2 R phos generated through incubation of Apo β 2 V 2 R phos with tenfold molar excess of carvedilol) exhibits a robust interaction with βarr1 in the presence of Fab30 as assessed by ELISA. Purified Fab30 was immobilized and then incubated with βarr1 and either Bias β 2 V 2 R phos or Act β 2 V 2 R phos . Formation of complex was detected using anti-FLAG M2 antibody. ( c ) Formation of βarr1 complex with Bias β 2 V 2 R phos in the presence of Fab30 as assessed by coimmunoprecipitation. The experiment was repeated three times with identical results and a representative image is shown. Quantification of the data is shown as bar graph. ( d ) Interaction of Bias β 2 AR phos +βarr1+ScFv30 and Act β 2 V 2 R phos +βarr1+ScFv30 complexes with inactive and ( e ) active ERK2. Purified ERK2 was immobilized followed by incubation with pre-formed complexes and detection using HRP-coupled anti-FLAG M2 antibody. ( f ) Interaction of Bias β 2 V 2 R phos with βarr1 does not lead to a detectable decrease in bimane fluorescence suggesting the lack of core interaction. Apo β 2 V 2 R phos was first incubated with tenfold molar excess of carvedilol or BI-167107 to obtain Bias β 2 V 2 R phos and Act β 2 V 2 R phos , respectively. Subsequently, these receptor preparations were incubated with bimane labelled βarr1 and Fab 30 to form a complex followed by fluorescence scanning in the wavelength range indicated on the graph. The data represent an average of three independent experiments. Data presented in b , d and e represent mean±s.e.m. of at least three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (** P

    Article Snippet: Codon optimized Fab30 open reading frame was synthesized (Genscript) based on published crystal structure (PDB ID: 4JQI) (ref. ), expressed and purified in M55244 strain of E. coli (purchased from American Type Culture Collection) .

    Techniques: Binding Assay, Activation Assay, Generated, Incubation, Enzyme-linked Immunosorbent Assay, Purification, Activated Clotting Time Assay, Fluorescence

    Validation of partially and fully engaged complexes by fluorescence spectroscopy. ( a ) Structural model of β 2 AR–β-arr1 complex deduced based on negative-stain electron microscopy, cross-linking experiments and hydrogen-deuterium exchange mass-spectrometry reveals finger loop of βarr1 as a key component of the core interaction. L 68 in the finger loop of βarr1 was changed to cysteine in a cysteine-less βarr1 and monobromobimane was attached to this cysteine by chemical coupling. Upon core interaction, bimane fluorescence intensity decreases either due to change in chemical environment or quenching by a tyrosine/tryptophan residue in the vicinity. ( b ) Functional validation of bimane labelled βarr1 by its interaction with purified β 2 V 2 R. Similar to wild-type βarr1, bimane labelled βarr1 also forms a complex with agonist occupied and phosphorylated β 2 V 2 R. The experiment was repeated twice with identical results and a representative image is shown. ( c ) Incubation of Act β 2 V 2 R phos but not Inact β 2 V 2 R phos with bimane labelled βarr1 leads to a decrease in bimane fluorescence. Considering equivalent physical interaction of Act β 2 V 2 R phos and Inact β 2 V 2 R phos (as presented in Fig. 1e,f ), bimane fluorescence data suggests that Act β 2 V 2 R phos engages the core interaction while the Inact β 2 V 2 R phos does not. These data suggest that Inact β 2 V 2 R phos +βarr1+Fab30 and Act β 2 V 2 R phos +βarr1+Fab30 complexes represent ‘tail only' and ‘fully' (tail+core) engaged complexes, respectively. ( d ) Bimane fluorescence at emission λ max as measured in c is presented as a bar graph. Data presented in d represent mean ±s.e.m. of three independent experiments analysed using one-way ANOVA with Bonferroni post-test (*** P

    Journal: Nature Communications

    Article Title: Functional competence of a partially engaged GPCR–β-arrestin complex

    doi: 10.1038/ncomms13416

    Figure Lengend Snippet: Validation of partially and fully engaged complexes by fluorescence spectroscopy. ( a ) Structural model of β 2 AR–β-arr1 complex deduced based on negative-stain electron microscopy, cross-linking experiments and hydrogen-deuterium exchange mass-spectrometry reveals finger loop of βarr1 as a key component of the core interaction. L 68 in the finger loop of βarr1 was changed to cysteine in a cysteine-less βarr1 and monobromobimane was attached to this cysteine by chemical coupling. Upon core interaction, bimane fluorescence intensity decreases either due to change in chemical environment or quenching by a tyrosine/tryptophan residue in the vicinity. ( b ) Functional validation of bimane labelled βarr1 by its interaction with purified β 2 V 2 R. Similar to wild-type βarr1, bimane labelled βarr1 also forms a complex with agonist occupied and phosphorylated β 2 V 2 R. The experiment was repeated twice with identical results and a representative image is shown. ( c ) Incubation of Act β 2 V 2 R phos but not Inact β 2 V 2 R phos with bimane labelled βarr1 leads to a decrease in bimane fluorescence. Considering equivalent physical interaction of Act β 2 V 2 R phos and Inact β 2 V 2 R phos (as presented in Fig. 1e,f ), bimane fluorescence data suggests that Act β 2 V 2 R phos engages the core interaction while the Inact β 2 V 2 R phos does not. These data suggest that Inact β 2 V 2 R phos +βarr1+Fab30 and Act β 2 V 2 R phos +βarr1+Fab30 complexes represent ‘tail only' and ‘fully' (tail+core) engaged complexes, respectively. ( d ) Bimane fluorescence at emission λ max as measured in c is presented as a bar graph. Data presented in d represent mean ±s.e.m. of three independent experiments analysed using one-way ANOVA with Bonferroni post-test (*** P

    Article Snippet: Codon optimized Fab30 open reading frame was synthesized (Genscript) based on published crystal structure (PDB ID: 4JQI) (ref. ), expressed and purified in M55244 strain of E. coli (purchased from American Type Culture Collection) .

    Techniques: Fluorescence, Spectroscopy, Staining, Electron Microscopy, Mass Spectrometry, Functional Assay, Purification, Incubation, Activated Clotting Time Assay

    Truncation of the third intracellular loop in β 2 V 2 R ablates core interaction with βarr1. ( a ) Cross-linking experiments and electron microscopy based structural model of β 2 V 2 R–βarr1 complex has identified the third intracellular loop of the β 2 V 2 R as prominent interface for core interaction through docking of the finger loop of βarr1. Residues that are identified to cross-link with each other in β 2 V 2 R–βarr1 complex are labelled and their side chains are highlighted as space fill model. ( b ) Cross-linking studies and X-ray crystal structure of rhodopsin-visual arrestin also displays the vicinity of the third intracellular loop in rhodopsin with the finger loop of visual arrestin. ( c ) Sequence alignment of β 2 V 2 R and β 2 V 2 R ΔICL3 (third intracellular loop truncated receptor) to highlight the deleted amino acids (Gly 238 -Lys 267 ) (red box). ( d ) Confocal microscopy of HEK-293 cells expressing either β 2 V 2 R or β 2 V 2 R ΔICL3 with β-arr1-YFP. Agonist stimulation leads to accumulation of endocytotic vesicles that indicates recruitment of βarr1 to activated receptor. Nuclear staining is shown using 4,6-diamidino-2-phenylindole. Compared with β 2 V 2 R, β 2 V 2 R ΔICL3 exhibits somewhat weaker recruitment of βarr1 as reflected by less punctate appearance. Scale bar, 10 μm. ( e ) Coimmunoprecipitation of β 2 V 2 R ΔICL3 with βarr1 expressed in HEK-293 cells further confirms the recruitment of βarr1 to the truncated receptor upon agonist stimulation. Cells were stimulated with agonist (Isoproterenol, 10 μM for 30 min at 37 °C) followed by cross-linking using dithiobis(succinimidyl-propionate) (1 mM for 30 min at room-temperature) and subsequently, receptor–βarr1 complex was coimmunoprecipitation using anti-FLAG antibody beads. ( f ) Assembly of β 2 V 2 R ΔICL3 +β-arr1+Fab30 complex as measured using ELISA approach and ( g ) coimmunoprecipitation experiment. Similar to β 2 V 2 R, β 2 V 2 R ΔICL3 also forms a stable complex with βarr1 in the presence of Fab30. ( h ) Quantification of β 2 V 2 R ΔICL3 –βarr1 complex formation as assessed by coimmunoprecipitation. ( i ) Bimane fluorescence spectroscopy on β 2 V 2 R ΔICL3 complex reveals the absence of fluorescence quenching even in the presence of agonist and thereby suggests the lack of core interaction. ( j ) Bimane fluorescence at emission λ max as measured in i is presented as bar graph. Data in f represents mean±s.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (*** P

    Journal: Nature Communications

    Article Title: Functional competence of a partially engaged GPCR–β-arrestin complex

    doi: 10.1038/ncomms13416

    Figure Lengend Snippet: Truncation of the third intracellular loop in β 2 V 2 R ablates core interaction with βarr1. ( a ) Cross-linking experiments and electron microscopy based structural model of β 2 V 2 R–βarr1 complex has identified the third intracellular loop of the β 2 V 2 R as prominent interface for core interaction through docking of the finger loop of βarr1. Residues that are identified to cross-link with each other in β 2 V 2 R–βarr1 complex are labelled and their side chains are highlighted as space fill model. ( b ) Cross-linking studies and X-ray crystal structure of rhodopsin-visual arrestin also displays the vicinity of the third intracellular loop in rhodopsin with the finger loop of visual arrestin. ( c ) Sequence alignment of β 2 V 2 R and β 2 V 2 R ΔICL3 (third intracellular loop truncated receptor) to highlight the deleted amino acids (Gly 238 -Lys 267 ) (red box). ( d ) Confocal microscopy of HEK-293 cells expressing either β 2 V 2 R or β 2 V 2 R ΔICL3 with β-arr1-YFP. Agonist stimulation leads to accumulation of endocytotic vesicles that indicates recruitment of βarr1 to activated receptor. Nuclear staining is shown using 4,6-diamidino-2-phenylindole. Compared with β 2 V 2 R, β 2 V 2 R ΔICL3 exhibits somewhat weaker recruitment of βarr1 as reflected by less punctate appearance. Scale bar, 10 μm. ( e ) Coimmunoprecipitation of β 2 V 2 R ΔICL3 with βarr1 expressed in HEK-293 cells further confirms the recruitment of βarr1 to the truncated receptor upon agonist stimulation. Cells were stimulated with agonist (Isoproterenol, 10 μM for 30 min at 37 °C) followed by cross-linking using dithiobis(succinimidyl-propionate) (1 mM for 30 min at room-temperature) and subsequently, receptor–βarr1 complex was coimmunoprecipitation using anti-FLAG antibody beads. ( f ) Assembly of β 2 V 2 R ΔICL3 +β-arr1+Fab30 complex as measured using ELISA approach and ( g ) coimmunoprecipitation experiment. Similar to β 2 V 2 R, β 2 V 2 R ΔICL3 also forms a stable complex with βarr1 in the presence of Fab30. ( h ) Quantification of β 2 V 2 R ΔICL3 –βarr1 complex formation as assessed by coimmunoprecipitation. ( i ) Bimane fluorescence spectroscopy on β 2 V 2 R ΔICL3 complex reveals the absence of fluorescence quenching even in the presence of agonist and thereby suggests the lack of core interaction. ( j ) Bimane fluorescence at emission λ max as measured in i is presented as bar graph. Data in f represents mean±s.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (*** P

    Article Snippet: Codon optimized Fab30 open reading frame was synthesized (Genscript) based on published crystal structure (PDB ID: 4JQI) (ref. ), expressed and purified in M55244 strain of E. coli (purchased from American Type Culture Collection) .

    Techniques: Electron Microscopy, Sequencing, Confocal Microscopy, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Fluorescence, Spectroscopy

    Core interaction is dispensable for recruitment of ERK2 MAP kinase. ( a ) An ELISA based approach to test the interaction of purified ERK2 with pre-formed β 2 V 2 R–βarr1-ScFv30 complex. Purified ERK2 (inactive or active) is immobilized on polystyrene surface followed by incubation with either the ‘tail only' engaged or ‘fully' engaged pre-formed complex. Interaction of ERK with the complex is visualized using HRP-coupled anti-FLAG M2 antibody as a read out of β 2 V 2 R retention on the plate. ( b ) Both ‘tail only' engaged ( Inact β 2 V 2 R phos +β-arr1+ScFv30) and ‘fully' engaged ( Act β 2 V 2 R phos +β-arr1+ScFv30) complexes interact with immobilized inactive (non-phosphorylated) ERK2. ( c ) Similar to inactive ERK2, phosphorylated ERK2 (that is, active) also interacts with both, the ‘tail only' engaged and ‘fully' engaged complexes. ( d ) A previously described conformationally selective nanobody (Nb6B9) against agonist bound β 2 AR conformation has an overlapping interface with the core interaction. Structural representation based on superimposition of crystal structure of agonist bound β 2 AR and nanobody Nb6B9 (PDB ID:4LDO) and electron microscopy based model of β 2 V 2 R–βarr1 complex. ( e ) Pre-incubation of Act β 2 V 2 R phos with purified Nb6B9 does not affect its physical interaction with βarr1. Purified Act β 2 V 2 R phos was first incubated with a threefold molar excess of Nb6B9 and subsequently used for the assembly of β 2 V 2 R–βarr1-Fab30 complex in ELISA format. ( f ) Pre-incubation of Act β 2 V 2 R phos with Nb6B9 abolishes bimane fluorescence quenching observed upon interaction with βarr1 suggesting that presence of Nb6B9 in Act β 2 AR phos +βarr1+Fab30 complex converts it to ‘tail only' engaged complex. ( g ) Interaction of inactive ERK2 and ( h ) active ERK2 with Nb6B9 stabilized ‘tail only' engaged complex as assessed by ELISA, further suggests that the core interaction is dispensable for ERK recruitment. Data presented in b , c , e , g and h represent mean±s.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test ( ** P

    Journal: Nature Communications

    Article Title: Functional competence of a partially engaged GPCR–β-arrestin complex

    doi: 10.1038/ncomms13416

    Figure Lengend Snippet: Core interaction is dispensable for recruitment of ERK2 MAP kinase. ( a ) An ELISA based approach to test the interaction of purified ERK2 with pre-formed β 2 V 2 R–βarr1-ScFv30 complex. Purified ERK2 (inactive or active) is immobilized on polystyrene surface followed by incubation with either the ‘tail only' engaged or ‘fully' engaged pre-formed complex. Interaction of ERK with the complex is visualized using HRP-coupled anti-FLAG M2 antibody as a read out of β 2 V 2 R retention on the plate. ( b ) Both ‘tail only' engaged ( Inact β 2 V 2 R phos +β-arr1+ScFv30) and ‘fully' engaged ( Act β 2 V 2 R phos +β-arr1+ScFv30) complexes interact with immobilized inactive (non-phosphorylated) ERK2. ( c ) Similar to inactive ERK2, phosphorylated ERK2 (that is, active) also interacts with both, the ‘tail only' engaged and ‘fully' engaged complexes. ( d ) A previously described conformationally selective nanobody (Nb6B9) against agonist bound β 2 AR conformation has an overlapping interface with the core interaction. Structural representation based on superimposition of crystal structure of agonist bound β 2 AR and nanobody Nb6B9 (PDB ID:4LDO) and electron microscopy based model of β 2 V 2 R–βarr1 complex. ( e ) Pre-incubation of Act β 2 V 2 R phos with purified Nb6B9 does not affect its physical interaction with βarr1. Purified Act β 2 V 2 R phos was first incubated with a threefold molar excess of Nb6B9 and subsequently used for the assembly of β 2 V 2 R–βarr1-Fab30 complex in ELISA format. ( f ) Pre-incubation of Act β 2 V 2 R phos with Nb6B9 abolishes bimane fluorescence quenching observed upon interaction with βarr1 suggesting that presence of Nb6B9 in Act β 2 AR phos +βarr1+Fab30 complex converts it to ‘tail only' engaged complex. ( g ) Interaction of inactive ERK2 and ( h ) active ERK2 with Nb6B9 stabilized ‘tail only' engaged complex as assessed by ELISA, further suggests that the core interaction is dispensable for ERK recruitment. Data presented in b , c , e , g and h represent mean±s.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test ( ** P

    Article Snippet: Codon optimized Fab30 open reading frame was synthesized (Genscript) based on published crystal structure (PDB ID: 4JQI) (ref. ), expressed and purified in M55244 strain of E. coli (purchased from American Type Culture Collection) .

    Techniques: Enzyme-linked Immunosorbent Assay, Purification, Incubation, Activated Clotting Time Assay, Electron Microscopy, Fluorescence

    Assembly of partially and fully engaged β 2 V 2 R–βarr1-Fab30 complex. ( a ) Schematic representation of biphasic GPCR–βarr interaction. βarr interacts with activated and phosphorylated GPCRs in a biphasic fashion where the first step is binding of βarr through the phosphorylated carboxyl terminus and the second step is the engagement of βarr with the 7TM core of the receptor. The receptor component is shown in grey, phosphorylated carboxyl terminus in yellow and βarr 1 in blue/magenta. ( b ) Schematic representation of an ELISA-based approach for in-vitro assembly of β 2 V 2 R–βarr1 complex. Purified Fab30 is immobilized on solid support as an anchor to capture the complex followed by incubation with purified β 2 V 2 R and βarr1. Formation of β 2 V 2 R–βarr1 complex is visualized using HRP-coupled anti-FLAG M2 antibody through detection of FLAG tagged β 2 V 2 R. ( c ) Fab 30 assisted in-vitro assembly of β 2 V 2 R–βarr1 complex. Agonist bound and phosphorylated β 2 V 2 R ( Act β 2 V 2 R phos ) forms a stable complex while inverse agonist bound and non-phosphorylated β 2 V 2 R ( Inact β 2 V 2 R non-phos ) does not exhibit any detectable complex formation. ( d ) An experimental set-up to assemble ‘tail only' engaged and ‘fully' engaged β 2 V 2 R–βarr1 complex in-vitro . β 2 V 2 R is coexpressed with GRK2 CAAX in cultured Sf 9 cells and 66 h post-infection, cells are stimulated with a low-affinity agonist (Isoproterenol) to trigger receptor phosphorylation. Subsequently, the receptor is purified by affinity chromatography and the ligand is washed off during purification to yield ligand free phosphorylated β 2 V 2 R ( Apo β 2 V 2 R phos ). Incubation with inverse agonist (carazolol) or high-affinity full agonist (BI-167107) yields Inact β 2 V 2 R phos and Act β 2 V 2 R phos , respectively. ( e ) Both, the Inact β 2 V 2 R phos and Act β 2 V 2 R phos form a stable complex with βarr1 as assessed by ELISA approach and potentially represent ‘tail only' and ‘fully' engaged complexes, respectively. ( f ) Formation of ‘tail only' engaged and ‘fully' engaged complexes as assessed by coimmunoprecipitation experiment. This experiment was repeated three times with identical results and a representative image is shown. Signals in c and e are normalized with Act β 2 V 2 R phos +βarr1+Fab30 condition as 100%. Data presented in c and e represent mean±s.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (*** P

    Journal: Nature Communications

    Article Title: Functional competence of a partially engaged GPCR–β-arrestin complex

    doi: 10.1038/ncomms13416

    Figure Lengend Snippet: Assembly of partially and fully engaged β 2 V 2 R–βarr1-Fab30 complex. ( a ) Schematic representation of biphasic GPCR–βarr interaction. βarr interacts with activated and phosphorylated GPCRs in a biphasic fashion where the first step is binding of βarr through the phosphorylated carboxyl terminus and the second step is the engagement of βarr with the 7TM core of the receptor. The receptor component is shown in grey, phosphorylated carboxyl terminus in yellow and βarr 1 in blue/magenta. ( b ) Schematic representation of an ELISA-based approach for in-vitro assembly of β 2 V 2 R–βarr1 complex. Purified Fab30 is immobilized on solid support as an anchor to capture the complex followed by incubation with purified β 2 V 2 R and βarr1. Formation of β 2 V 2 R–βarr1 complex is visualized using HRP-coupled anti-FLAG M2 antibody through detection of FLAG tagged β 2 V 2 R. ( c ) Fab 30 assisted in-vitro assembly of β 2 V 2 R–βarr1 complex. Agonist bound and phosphorylated β 2 V 2 R ( Act β 2 V 2 R phos ) forms a stable complex while inverse agonist bound and non-phosphorylated β 2 V 2 R ( Inact β 2 V 2 R non-phos ) does not exhibit any detectable complex formation. ( d ) An experimental set-up to assemble ‘tail only' engaged and ‘fully' engaged β 2 V 2 R–βarr1 complex in-vitro . β 2 V 2 R is coexpressed with GRK2 CAAX in cultured Sf 9 cells and 66 h post-infection, cells are stimulated with a low-affinity agonist (Isoproterenol) to trigger receptor phosphorylation. Subsequently, the receptor is purified by affinity chromatography and the ligand is washed off during purification to yield ligand free phosphorylated β 2 V 2 R ( Apo β 2 V 2 R phos ). Incubation with inverse agonist (carazolol) or high-affinity full agonist (BI-167107) yields Inact β 2 V 2 R phos and Act β 2 V 2 R phos , respectively. ( e ) Both, the Inact β 2 V 2 R phos and Act β 2 V 2 R phos form a stable complex with βarr1 as assessed by ELISA approach and potentially represent ‘tail only' and ‘fully' engaged complexes, respectively. ( f ) Formation of ‘tail only' engaged and ‘fully' engaged complexes as assessed by coimmunoprecipitation experiment. This experiment was repeated three times with identical results and a representative image is shown. Signals in c and e are normalized with Act β 2 V 2 R phos +βarr1+Fab30 condition as 100%. Data presented in c and e represent mean±s.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (*** P

    Article Snippet: Codon optimized Fab30 open reading frame was synthesized (Genscript) based on published crystal structure (PDB ID: 4JQI) (ref. ), expressed and purified in M55244 strain of E. coli (purchased from American Type Culture Collection) .

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, In Vitro, Purification, Incubation, Activated Clotting Time Assay, Cell Culture, Infection, Affinity Chromatography

    Structures of S. pombe Lsm2-8 bound to pentauridylate with a 2’,3’ OH or 2’,3’ cyclic phosphate. A) Overview of Lsm2-8 bound to “unprocessed” U6 snRNA 3’ terminus. B) Overview of Lsm2-8 bound to “mature” U6 snRNA 3’ terminus. C) Detail of Lsm2-8 interface with “unprocessed” RNA. The 3’ terminal uridine is disordered and thus not visible in the final electron density maps. D) In contrast, the mature U6 3’ end, with a 2’,3′ cyclic phosphate, shows electron density for the terminal nucleotide. E) The Sm-like pocket in Lsm3 binds RNA as observed previously in other Lsm2-8 complexes from S. cerevisiae . F) In contrast, the 3′ uridine cyclic phosphate has a unique binding mechanism relative to the other four uridines, including a stacking interaction with the C-terminal histidine of Lsm8.

    Journal: bioRxiv

    Article Title: Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

    doi: 10.1101/2020.04.22.055376

    Figure Lengend Snippet: Structures of S. pombe Lsm2-8 bound to pentauridylate with a 2’,3’ OH or 2’,3’ cyclic phosphate. A) Overview of Lsm2-8 bound to “unprocessed” U6 snRNA 3’ terminus. B) Overview of Lsm2-8 bound to “mature” U6 snRNA 3’ terminus. C) Detail of Lsm2-8 interface with “unprocessed” RNA. The 3’ terminal uridine is disordered and thus not visible in the final electron density maps. D) In contrast, the mature U6 3’ end, with a 2’,3′ cyclic phosphate, shows electron density for the terminal nucleotide. E) The Sm-like pocket in Lsm3 binds RNA as observed previously in other Lsm2-8 complexes from S. cerevisiae . F) In contrast, the 3′ uridine cyclic phosphate has a unique binding mechanism relative to the other four uridines, including a stacking interaction with the C-terminal histidine of Lsm8.

    Article Snippet: Production of recombinant Lsm2-8 Codon optimized open reading frames (Genscript) for S. pombe Lsm proteins were cloned into modified variants of the pQLink expression system ( ).

    Techniques: Binding Assay

    The elongated C-terminal region of Lsm1 in the S. pombe Lsm1-7 ring attenuates RNA binding. A) Structure of the S. cerevisiae Lsm1-7 ring in the absence of bound RNA ( 11 ). The C-terminal region of Lsm1 crosses the distal face of the ring, occluding the central pore. B) Superposition of U6 snRNA from the homologous S. cerevisiae Lsm2-8 ring with the S. cerevisiae Lsm1-7 ring. The alignment in this figure was achieved by superposition of Lsm2, Lsm3 and Lsm6 components of the rings, which share a pairwise r.m.s.d of 0.5 Å. A steric clash is visible between the 3′ uridine phosphate and the C-terminal region of Lsm1. C) In vitro fluorescence polarization binding assays show that S. pombe Lsm1-7 tightly binds to polyuridylate tracts without accessory proteins, like Pat1. In contrast, S. pombe Lsm1-7 lacks detectible affinity for polyadenylate. D) Designed truncations of the Lsm1 C-terminus. Lsm1 is colored blue to red from the N and C termini, respectively, and regions selected for truncation are annotated in the figure, resulting in truncation of the last 12 residues that fold back into the pore, or truncation of the entire helical region that spans the distal face of the ring. E) Binding assays showing that deletion of the Lsm1 C-terminal region generally enhances binding affinity for RNAs that harbor polyuridine tracts, and the relative enhancement is greatest for the weakest binding RNAs. F) A strong preference for an adenosine 3’ terminus over a uridine cyclic phosphate 3’ terminus is diminished upon deletion of the C-terminal 12 residues of Lsm1.

    Journal: bioRxiv

    Article Title: Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

    doi: 10.1101/2020.04.22.055376

    Figure Lengend Snippet: The elongated C-terminal region of Lsm1 in the S. pombe Lsm1-7 ring attenuates RNA binding. A) Structure of the S. cerevisiae Lsm1-7 ring in the absence of bound RNA ( 11 ). The C-terminal region of Lsm1 crosses the distal face of the ring, occluding the central pore. B) Superposition of U6 snRNA from the homologous S. cerevisiae Lsm2-8 ring with the S. cerevisiae Lsm1-7 ring. The alignment in this figure was achieved by superposition of Lsm2, Lsm3 and Lsm6 components of the rings, which share a pairwise r.m.s.d of 0.5 Å. A steric clash is visible between the 3′ uridine phosphate and the C-terminal region of Lsm1. C) In vitro fluorescence polarization binding assays show that S. pombe Lsm1-7 tightly binds to polyuridylate tracts without accessory proteins, like Pat1. In contrast, S. pombe Lsm1-7 lacks detectible affinity for polyadenylate. D) Designed truncations of the Lsm1 C-terminus. Lsm1 is colored blue to red from the N and C termini, respectively, and regions selected for truncation are annotated in the figure, resulting in truncation of the last 12 residues that fold back into the pore, or truncation of the entire helical region that spans the distal face of the ring. E) Binding assays showing that deletion of the Lsm1 C-terminal region generally enhances binding affinity for RNAs that harbor polyuridine tracts, and the relative enhancement is greatest for the weakest binding RNAs. F) A strong preference for an adenosine 3’ terminus over a uridine cyclic phosphate 3’ terminus is diminished upon deletion of the C-terminal 12 residues of Lsm1.

    Article Snippet: Production of recombinant Lsm2-8 Codon optimized open reading frames (Genscript) for S. pombe Lsm proteins were cloned into modified variants of the pQLink expression system ( ).

    Techniques: RNA Binding Assay, In Vitro, Fluorescence, Binding Assay

    Comparison of how 3’ processed U6 RNA is recognized by S. cerevisiae and S. pombe Lsm2-8. A) S. pombe Lsm2-8 binds the 3′ uridine cyclic phosphate in the middle of the ring through non-Sm contacts with Lsm8 and Lsm3. B) The C-terminal tail of Lsm8 in S. cerevisiae is elongated and extends past the terminal uridine-phosphate. In lieu of direct interaction with 3′ uridine-phosphate, the tail of Lsm8 in S. cerevisiae interacts with the phosphate through long range electrostatics. C,D) Detailed comparisons of how the cyclic and non-cyclic phosphates are coordinated by the S. pombe and S. cerevisiae Lsm2-8 rings, respectively. Lsm3-Arg27 directly coordinates the cyclic phosphate in S. pombe .

    Journal: bioRxiv

    Article Title: Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

    doi: 10.1101/2020.04.22.055376

    Figure Lengend Snippet: Comparison of how 3’ processed U6 RNA is recognized by S. cerevisiae and S. pombe Lsm2-8. A) S. pombe Lsm2-8 binds the 3′ uridine cyclic phosphate in the middle of the ring through non-Sm contacts with Lsm8 and Lsm3. B) The C-terminal tail of Lsm8 in S. cerevisiae is elongated and extends past the terminal uridine-phosphate. In lieu of direct interaction with 3′ uridine-phosphate, the tail of Lsm8 in S. cerevisiae interacts with the phosphate through long range electrostatics. C,D) Detailed comparisons of how the cyclic and non-cyclic phosphates are coordinated by the S. pombe and S. cerevisiae Lsm2-8 rings, respectively. Lsm3-Arg27 directly coordinates the cyclic phosphate in S. pombe .

    Article Snippet: Production of recombinant Lsm2-8 Codon optimized open reading frames (Genscript) for S. pombe Lsm proteins were cloned into modified variants of the pQLink expression system ( ).

    Techniques:

    The structure of S. pombe Lsm1-7 bound to RNA explains the preference for a 3’ terminal purine. A) Overview of Lsm1-7 bound to UUUUA. B) Overview of Lsm1-7 bound to AUUUUG. C) RNA-binding interface of Lsm1-7 bound to UUUUA, showing that four uracil bases bind in the same manner as in the Lsm2-8 complex, while the adenosine binds into non-Sm pocket in Lsm5. D) Lsm1-7 bound to AUUUUG, showing a similar binding mechanism as in panel C. E,F) Detailed view of the non-Sm Lsm5 binding pocket occupied by adenine or guanine. In both cases, the 3′ purine is coordinated through hydrogen bonding with Lsm5-Asn66 and stacking with Lsm5-Asn68.

    Journal: bioRxiv

    Article Title: Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

    doi: 10.1101/2020.04.22.055376

    Figure Lengend Snippet: The structure of S. pombe Lsm1-7 bound to RNA explains the preference for a 3’ terminal purine. A) Overview of Lsm1-7 bound to UUUUA. B) Overview of Lsm1-7 bound to AUUUUG. C) RNA-binding interface of Lsm1-7 bound to UUUUA, showing that four uracil bases bind in the same manner as in the Lsm2-8 complex, while the adenosine binds into non-Sm pocket in Lsm5. D) Lsm1-7 bound to AUUUUG, showing a similar binding mechanism as in panel C. E,F) Detailed view of the non-Sm Lsm5 binding pocket occupied by adenine or guanine. In both cases, the 3′ purine is coordinated through hydrogen bonding with Lsm5-Asn66 and stacking with Lsm5-Asn68.

    Article Snippet: Production of recombinant Lsm2-8 Codon optimized open reading frames (Genscript) for S. pombe Lsm proteins were cloned into modified variants of the pQLink expression system ( ).

    Techniques: RNA Binding Assay, Binding Assay

    3’-processing of U6 snRNA alters its recognition by S. pombe Lsm2-8. A) Processing of U6 by Usb1 leaves a 3′ cyclic phosphate group on U6, denoted as “ > p”. B) In vitro binding assays show that S. pombe Lsm2-8 preferentially binds RNA that has been processed by Usb1. All binding curves and K d determinations in this work were performed with a restrained Hill coefficient of 1. For reference, the non-restrained Hill coefficients, which are close to 1, are shown in the figure.

    Journal: bioRxiv

    Article Title: Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

    doi: 10.1101/2020.04.22.055376

    Figure Lengend Snippet: 3’-processing of U6 snRNA alters its recognition by S. pombe Lsm2-8. A) Processing of U6 by Usb1 leaves a 3′ cyclic phosphate group on U6, denoted as “ > p”. B) In vitro binding assays show that S. pombe Lsm2-8 preferentially binds RNA that has been processed by Usb1. All binding curves and K d determinations in this work were performed with a restrained Hill coefficient of 1. For reference, the non-restrained Hill coefficients, which are close to 1, are shown in the figure.

    Article Snippet: Production of recombinant Lsm2-8 Codon optimized open reading frames (Genscript) for S. pombe Lsm proteins were cloned into modified variants of the pQLink expression system ( ).

    Techniques: In Vitro, Binding Assay

    Structural details of S. pombe Lsm2-8 with RNA. A) Overview of unprocessed pentauridylate bound to Lsm2-8. Only the bridging phosphodiester between the last and penultimate uridine nucleotides is clearly visible in the electron density maps. B) Denaturing urea PAGE gels showing that prolonged incubation with Lsm2-8 does not result in cleavage and shortening of diol-terminated RNA, and as such the observed terminal phosphate in panel A is not likely to be a product of hydrolysis. C,D) Simulated annealing omit maps for RNA bound to Lsm2-8. Depicted maps are of form m F o - D F c , and are contoured at 1 r.m.s.d. E) Crystal packing of Lsm2-8 complexes in space group P 2 1 2 1 2 is bridged by an unidentified buffer component that is putatively a contaminant within the pentaerythritol propoxylate (5/4 PO/OH) precipitant used for crystal growth. The depicted map is identical to that in panels C and D. F) Two Lsm2-8 complexes are present in the crystallographic asymmetric unit, which vary significantly in their local atomic displacement parameters (colored blue to red), especially around the Lsm5 subunits. Representative local 2 m F o - D F c maps at 1 r.m.s.d. are shown in the inserts. G) The poor density in one of the Lsm2-8 rings can be attributed to a lack of substantial crystal packing contacts in one copy of Lsm2-8. An asterisk denotes the same regions of poor main chain density in panels F and G.

    Journal: bioRxiv

    Article Title: Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

    doi: 10.1101/2020.04.22.055376

    Figure Lengend Snippet: Structural details of S. pombe Lsm2-8 with RNA. A) Overview of unprocessed pentauridylate bound to Lsm2-8. Only the bridging phosphodiester between the last and penultimate uridine nucleotides is clearly visible in the electron density maps. B) Denaturing urea PAGE gels showing that prolonged incubation with Lsm2-8 does not result in cleavage and shortening of diol-terminated RNA, and as such the observed terminal phosphate in panel A is not likely to be a product of hydrolysis. C,D) Simulated annealing omit maps for RNA bound to Lsm2-8. Depicted maps are of form m F o - D F c , and are contoured at 1 r.m.s.d. E) Crystal packing of Lsm2-8 complexes in space group P 2 1 2 1 2 is bridged by an unidentified buffer component that is putatively a contaminant within the pentaerythritol propoxylate (5/4 PO/OH) precipitant used for crystal growth. The depicted map is identical to that in panels C and D. F) Two Lsm2-8 complexes are present in the crystallographic asymmetric unit, which vary significantly in their local atomic displacement parameters (colored blue to red), especially around the Lsm5 subunits. Representative local 2 m F o - D F c maps at 1 r.m.s.d. are shown in the inserts. G) The poor density in one of the Lsm2-8 rings can be attributed to a lack of substantial crystal packing contacts in one copy of Lsm2-8. An asterisk denotes the same regions of poor main chain density in panels F and G.

    Article Snippet: Production of recombinant Lsm2-8 Codon optimized open reading frames (Genscript) for S. pombe Lsm proteins were cloned into modified variants of the pQLink expression system ( ).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation

    Proposed model for Pat1-regulated gating of the Lsm1-7 RNA-binding pore. A) Relative orientations of RNA, Lsm1-7 and Pat1 proteins, derived from structures of S. pombe Lsm1-7 with RNA and S. cerevisiae Lsm1-7 with the C-terminal domain of Pat1 ( 11 ). B) Model for RNA binding specificity in Lsm rings. The Lsm2-8 ring contains a uridine-cyclic phosphate binding site that includes the C-terminus of Lsm8 and endows specificity for Usb1 processed U6 snRNA. C) In contrast, the C-terminal region of Lsm1 in the Lsm1-7 ring antagonizes association of the ring with uridine-cyclic phosphate terminated RNA, while allowing association of the ring with 3′ monopurinated polyuridylate tracts. D) Deletion of the C-terminal region of Lsm1 allows the ring to associate with a broader range of RNAs. In vivo , the role of Lsm1 truncation may be mimicked by displacement of the C-terminus by association of the ring with the Pat1 co-factor. It remains to be seen if RNA is capable of threading all the way through the pore of the RNA, as depicted here, or rather enters and exits the pore on the proximal face alone.

    Journal: bioRxiv

    Article Title: Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

    doi: 10.1101/2020.04.22.055376

    Figure Lengend Snippet: Proposed model for Pat1-regulated gating of the Lsm1-7 RNA-binding pore. A) Relative orientations of RNA, Lsm1-7 and Pat1 proteins, derived from structures of S. pombe Lsm1-7 with RNA and S. cerevisiae Lsm1-7 with the C-terminal domain of Pat1 ( 11 ). B) Model for RNA binding specificity in Lsm rings. The Lsm2-8 ring contains a uridine-cyclic phosphate binding site that includes the C-terminus of Lsm8 and endows specificity for Usb1 processed U6 snRNA. C) In contrast, the C-terminal region of Lsm1 in the Lsm1-7 ring antagonizes association of the ring with uridine-cyclic phosphate terminated RNA, while allowing association of the ring with 3′ monopurinated polyuridylate tracts. D) Deletion of the C-terminal region of Lsm1 allows the ring to associate with a broader range of RNAs. In vivo , the role of Lsm1 truncation may be mimicked by displacement of the C-terminus by association of the ring with the Pat1 co-factor. It remains to be seen if RNA is capable of threading all the way through the pore of the RNA, as depicted here, or rather enters and exits the pore on the proximal face alone.

    Article Snippet: Production of recombinant Lsm2-8 Codon optimized open reading frames (Genscript) for S. pombe Lsm proteins were cloned into modified variants of the pQLink expression system ( ).

    Techniques: RNA Binding Assay, Derivative Assay, Binding Assay, In Vivo

    Production of recombinant S. pombe Lsm rings that can bind RNA and form macromolecular complexes. A) SDS-PAGE analysis of Lsm2-8 and Lsm1-7 proteins made by the pQLink system ( 36 ) in E. coli and purified with the use of multiple, cleavable affinity tags. Lsm4 appears as a very close doublet, either due to incomplete denaturation on SDS PAGE or partial proteolysis of its C-terminus which has no visible electron density in the crystal structures reported here and is predicted to be a region of low complexity. All masses for all Lsm proteins were confirmed by mass spectrometry and no proteolytic fragments of Lsm4 were detected by mass spectrometry. The Lsm7 subunit in Lsm2-8 (lane 1) retains a C-terminal oligohistidine tag that is not present in the final Lsm1-7 samples (lanes 2-4). B) S. pombe Lsm2-8 can form recombinant U6 snRNPs after addition of Prp24 and U6 snRNA.

    Journal: bioRxiv

    Article Title: Molecular basis for the distinct cellular functions of the Lsm1-7 and Lsm2-8 complexes

    doi: 10.1101/2020.04.22.055376

    Figure Lengend Snippet: Production of recombinant S. pombe Lsm rings that can bind RNA and form macromolecular complexes. A) SDS-PAGE analysis of Lsm2-8 and Lsm1-7 proteins made by the pQLink system ( 36 ) in E. coli and purified with the use of multiple, cleavable affinity tags. Lsm4 appears as a very close doublet, either due to incomplete denaturation on SDS PAGE or partial proteolysis of its C-terminus which has no visible electron density in the crystal structures reported here and is predicted to be a region of low complexity. All masses for all Lsm proteins were confirmed by mass spectrometry and no proteolytic fragments of Lsm4 were detected by mass spectrometry. The Lsm7 subunit in Lsm2-8 (lane 1) retains a C-terminal oligohistidine tag that is not present in the final Lsm1-7 samples (lanes 2-4). B) S. pombe Lsm2-8 can form recombinant U6 snRNPs after addition of Prp24 and U6 snRNA.

    Article Snippet: Production of recombinant Lsm2-8 Codon optimized open reading frames (Genscript) for S. pombe Lsm proteins were cloned into modified variants of the pQLink expression system ( ).

    Techniques: Recombinant, SDS Page, Purification, Mass Spectrometry

    Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, UL89, or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 ORF or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.

    Journal: Journal of Virology

    Article Title: Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation

    doi: 10.1128/JVI.02384-16

    Figure Lengend Snippet: Reduced amounts of the terminase subunits in cells transfected with HCMV BAC genomes in which either UL51, UL56, UL89, or UL52 is disrupted. (A, left) RPE-1 cells were adenofected with the indicated BACs or were mock transfected. On day 4 posttransfection, cells were harvested, whole-cell lysates were prepared, and immunoblot analysis was performed with the antibodies indicated. The HCMV late protein pUL52 and the early protein pUL44 served as controls for both transfection efficiency and viral gene expression, and GAPDH was used as a loading control. The asterisks denote unspecific reactivity with a cellular protein. (Right) Immunoblot signals of three independent experiments (means ± standard deviations [SD] [error bars]) were quantified with ImageJ software using membranes exposed for a few seconds only. For normalization, UL51, UL56, and UL89 protein levels of cells transfected with the parental BAC pHG-UL51-SF/HA were set at 100%. (B) RPE-1 cells were adenofected with the BAC genome pHG-ΔUL52 carrying a deletion within the UL52 ORF or with the parental BAC pHG and analyzed by immunoblotting as in panel A with the antibodies indicated (HCMV major capsid [late] protein [MCP]). Quantification of signals was done as described above for panel A. (C) RPE-1 cells adenofected with the indicated HCMV BACs were used for preparation of total RNA on day 4 posttransfection. UL51, UL56, UL89, and UL52 transcript levels were determined by quantitative RT-PCR, relative RNA levels were calculated using UL52 as the internal control and normalized to the values for cells transfected with the parental BAC pHG-UL51-SF/HA. Data are representative of two independent experiments. (D and E) Interactions between the terminase subunits in RPE-1 cells adenofected with the indicated BACs. pUL51-SF/HA was pulled down using Strep-Tactin Sepharose (D), or pUL56 and pUL89 were immunoprecipitated (IP) with specific antibodies from cell lysates prepared on day 4 posttransfection (E). Eluted proteins were analyzed by immunoblotting using antibodies directed against the HA tag (for pUL51), pUL56, or pUL89. IgG heavy chains (HC) and light chains (LC) served as controls.

    Article Snippet: The resulting PCR product was inserted via In-Fusion cloning (Clontech) into pcDNA3.1(+) using the EcoRI and NotI sites. pcDNA-UL89 was constructed likewise by PCR amplification of human codon-optimized ORF UL89 (GeneArt, Germany) using primers 5_UL89codopt (codopt stands for codon optimized) (5′-GGCCGCATTTGCGAATTCGCCACCATGCTGAGAGGCGATAGCGCCGCC-3′) and 3′UL89codopt (5′-GGGCCCTCTAGCTCGAGTCATCAGGACACCCGGAACCG-3′) followed by In-Fusion cloning into pcDNA3.1(+) via the EcoRI and XhoI sites.

    Techniques: Transfection, BAC Assay, Expressing, Software, Quantitative RT-PCR, Immunoprecipitation

    Density gradient centrifugation of SV40 and MCVSyn particles. Optiprep™ gradient centrifugation was performed with cell lysates from CV1 (A) and H1299 (B) cells 4d after transfection with viral DNA. 15×250 µl fractions were collected (fraction 1 represents the fraction with the highest density and fraction 15 represents the lowest density fraction). (A) Left panel: Real time PCR of micrococcal nuclease treated fractions was performed using SV40 VP1 primer sequences. 20 µl of each gradient fraction was loaded on a 10% SDS-page followed immunoblotting using anti-VP1 serum. Right panel: Negative EM staining of SV40 particles identified in fraction 9. (B) Left panel: Real time PCR results of H1299 MCVSyn gradient fractions after micrococcal nuclease treatment using MCPyV VP1-specific primers. Right panel: Negative EM staining of particles identified in fractions 10 and 6.

    Journal: PLoS ONE

    Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

    doi: 10.1371/journal.pone.0029112

    Figure Lengend Snippet: Density gradient centrifugation of SV40 and MCVSyn particles. Optiprep™ gradient centrifugation was performed with cell lysates from CV1 (A) and H1299 (B) cells 4d after transfection with viral DNA. 15×250 µl fractions were collected (fraction 1 represents the fraction with the highest density and fraction 15 represents the lowest density fraction). (A) Left panel: Real time PCR of micrococcal nuclease treated fractions was performed using SV40 VP1 primer sequences. 20 µl of each gradient fraction was loaded on a 10% SDS-page followed immunoblotting using anti-VP1 serum. Right panel: Negative EM staining of SV40 particles identified in fraction 9. (B) Left panel: Real time PCR results of H1299 MCVSyn gradient fractions after micrococcal nuclease treatment using MCPyV VP1-specific primers. Right panel: Negative EM staining of particles identified in fractions 10 and 6.

    Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

    Techniques: Gradient Centrifugation, Transfection, Real-time Polymerase Chain Reaction, SDS Page, Staining

    Subcellular localization of LT-Ag and VP1 protein in cells transfected with religated viral DNA. (A) Double staining of CV-1 cells transfected with SV40 viral DNA. 4d p.t. the cells were fixed, and VP1 was detected with a polyclonal anti-VP1 antibody. LT-Ag was visualized with the monoclonal anti-LT antibody Pab419. Z-stack pictures were taken using confocal microscopy. Each picture represents an individual Z-stack. VP1 staining was observed primarily in speckles close to or at the nuclear membrane. LT-Ag staining was observed throughout the nucleoplasm with the nucleoli excluded. In some cells granular LT-Ag staining was observed. The panel on the lower right represents a 3× zoomed picture of a CV1 transfected cell with the two channels merged. Double staining of Merkel cell polyomavirus VP1 and LT-Ag in H1299 cells (B) and PFSK-1 cells (C) 4d p.t. reveals inner peripheral nuclear localization of MCVSyn VP1 protein. VP1 was visualized with a polyclonal anti-VP1 serum and anti-rabbit FITC, while LT-Ag was visualized with the monoclonal antibody Cm2B4 specifically recognizing MCPyV LT-Ag. 40 Z-stack pictures were taken scanning through the cells using a 63× magnification and 2fold zoom on a confocal microscope. The picture shown represents an individual image from the center of a Z-stack.

    Journal: PLoS ONE

    Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

    doi: 10.1371/journal.pone.0029112

    Figure Lengend Snippet: Subcellular localization of LT-Ag and VP1 protein in cells transfected with religated viral DNA. (A) Double staining of CV-1 cells transfected with SV40 viral DNA. 4d p.t. the cells were fixed, and VP1 was detected with a polyclonal anti-VP1 antibody. LT-Ag was visualized with the monoclonal anti-LT antibody Pab419. Z-stack pictures were taken using confocal microscopy. Each picture represents an individual Z-stack. VP1 staining was observed primarily in speckles close to or at the nuclear membrane. LT-Ag staining was observed throughout the nucleoplasm with the nucleoli excluded. In some cells granular LT-Ag staining was observed. The panel on the lower right represents a 3× zoomed picture of a CV1 transfected cell with the two channels merged. Double staining of Merkel cell polyomavirus VP1 and LT-Ag in H1299 cells (B) and PFSK-1 cells (C) 4d p.t. reveals inner peripheral nuclear localization of MCVSyn VP1 protein. VP1 was visualized with a polyclonal anti-VP1 serum and anti-rabbit FITC, while LT-Ag was visualized with the monoclonal antibody Cm2B4 specifically recognizing MCPyV LT-Ag. 40 Z-stack pictures were taken scanning through the cells using a 63× magnification and 2fold zoom on a confocal microscope. The picture shown represents an individual image from the center of a Z-stack.

    Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

    Techniques: Transfection, Double Staining, Confocal Microscopy, Staining, Microscopy

    Retrotransposition driven by full-length L1 elements containing mutations in putative phosphorylation sites within ORF2. a Alu retrotransposition: Full-length L1 elements containing the indicated putative phosphorylation mutations within the ORF2 sequence were used to drive Alu retrotransposition in HeLa cells, as previously described [ 3 ]. L1 is the functional element and L1 EN- is a non-functional element containing a mutation in the ORF2 endonuclease domain (D205A). Control indicates cells transfected with an empty vector and the Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by Neo R colonies (Y-axis). b L1 retrotransposition: Full-length L1 elements containing the indicated putative phosphorylation mutations within the ORF2 sequence were used in an L1 retrotransposition assay in HeLa cells, as previously described [ 5 , 56 ]. L1 is the functional element and control indicates cells transfected with an empty plasmid. The graph depicts the relative number of L1 retrotransposition events as represented by Neo R colonies (Y-axis)

    Journal: Mobile DNA

    Article Title: The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

    doi: 10.1186/s13100-016-0064-x

    Figure Lengend Snippet: Retrotransposition driven by full-length L1 elements containing mutations in putative phosphorylation sites within ORF2. a Alu retrotransposition: Full-length L1 elements containing the indicated putative phosphorylation mutations within the ORF2 sequence were used to drive Alu retrotransposition in HeLa cells, as previously described [ 3 ]. L1 is the functional element and L1 EN- is a non-functional element containing a mutation in the ORF2 endonuclease domain (D205A). Control indicates cells transfected with an empty vector and the Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by Neo R colonies (Y-axis). b L1 retrotransposition: Full-length L1 elements containing the indicated putative phosphorylation mutations within the ORF2 sequence were used in an L1 retrotransposition assay in HeLa cells, as previously described [ 5 , 56 ]. L1 is the functional element and control indicates cells transfected with an empty plasmid. The graph depicts the relative number of L1 retrotransposition events as represented by Neo R colonies (Y-axis)

    Article Snippet: ORF2 putative phosphorylation mutants Mutations were introduced into a previously reported [ ] codon-optimized ORF2 expression plasmid (pBudCE4.1, Invitrogen) using the QuikChange Site-Directed Mutagenesis kit (Stratagene) per the manufacturer’s protocol.

    Techniques: Sequencing, Functional Assay, Mutagenesis, Transfection, Plasmid Preparation

    Expression of ORF2p containing mutations in selected putative phosphorylation sites outside of the endonuclease domain. Top panel: Representative western blot analysis of total cell lysates harvested from HeLa cells transfected with the indicated ORF2 putative phosphorylation mutant constructs. ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control lanes indicate cells transfected with an empty vector. Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 protein. Bottom panel: Western blot quantitation. For each sample, the signal detected for ORF2p was normalized to the total protein load. These relative numbers were expressed as a proportion of the relative number detected from the functional ORF2p. Asterisk denotes a significant difference in the steady-state levels relative to the functional ORF2p ( t -test, P ≤ 0.05)

    Journal: Mobile DNA

    Article Title: The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

    doi: 10.1186/s13100-016-0064-x

    Figure Lengend Snippet: Expression of ORF2p containing mutations in selected putative phosphorylation sites outside of the endonuclease domain. Top panel: Representative western blot analysis of total cell lysates harvested from HeLa cells transfected with the indicated ORF2 putative phosphorylation mutant constructs. ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control lanes indicate cells transfected with an empty vector. Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 protein. Bottom panel: Western blot quantitation. For each sample, the signal detected for ORF2p was normalized to the total protein load. These relative numbers were expressed as a proportion of the relative number detected from the functional ORF2p. Asterisk denotes a significant difference in the steady-state levels relative to the functional ORF2p ( t -test, P ≤ 0.05)

    Article Snippet: ORF2 putative phosphorylation mutants Mutations were introduced into a previously reported [ ] codon-optimized ORF2 expression plasmid (pBudCE4.1, Invitrogen) using the QuikChange Site-Directed Mutagenesis kit (Stratagene) per the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Transfection, Mutagenesis, Construct, Functional Assay, Plasmid Preparation, Generated, Quantitation Assay

    Expression and detection of ORF2 proteins containing mutations in putative phosphorylation sites. Top panel: Representative western blot analysis of total cell lysates harvested from HeLa cells transfected with the indicated ORF2 putative phosphorylation mutant constructs. ORF2 is the functional protein and ORF2 RT- is a non-functional protein containing a mutation in the reverse transcriptase (D702A) domain. Control lanes indicate cells transfected with an empty vector. Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 protein. Bottom panel: Western blot quantitation. For each sample, the signal detected for ORF2p was normalized to the total protein load. These relative numbers were expressed as a proportion of the relative number detected from the functional ORF2p. Asterisk denotes a significant difference in the steady-state levels relative to the functional ORF2p ( t -test, P ≤ 0.05)

    Journal: Mobile DNA

    Article Title: The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

    doi: 10.1186/s13100-016-0064-x

    Figure Lengend Snippet: Expression and detection of ORF2 proteins containing mutations in putative phosphorylation sites. Top panel: Representative western blot analysis of total cell lysates harvested from HeLa cells transfected with the indicated ORF2 putative phosphorylation mutant constructs. ORF2 is the functional protein and ORF2 RT- is a non-functional protein containing a mutation in the reverse transcriptase (D702A) domain. Control lanes indicate cells transfected with an empty vector. Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 protein. Bottom panel: Western blot quantitation. For each sample, the signal detected for ORF2p was normalized to the total protein load. These relative numbers were expressed as a proportion of the relative number detected from the functional ORF2p. Asterisk denotes a significant difference in the steady-state levels relative to the functional ORF2p ( t -test, P ≤ 0.05)

    Article Snippet: ORF2 putative phosphorylation mutants Mutations were introduced into a previously reported [ ] codon-optimized ORF2 expression plasmid (pBudCE4.1, Invitrogen) using the QuikChange Site-Directed Mutagenesis kit (Stratagene) per the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Transfection, Mutagenesis, Construct, Functional Assay, Plasmid Preparation, Generated, Quantitation Assay

    Analysis of select putative phosphorylation sites outside of the endonuclease domain. a Alu retrotransposition: ORF2 proteins containing mutations in the indicated putative phosphorylation sites were used to drive Alu retrotransposition in HeLa cells, as previously described [ 3 ]. ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by Neo R colonies (Y-axis). Asterisks indicate a statistically significant difference in Alu retrotransposition compared to ORF2 ( t -test, P ≤ 0.05). b Acute toxicity: HeLa cells were cotransfected with a Neo R expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. c Acute toxicity: 293 cells were cotransfected with a Neo R expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. In both panels b and c , ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the Neo R expression vector. Colony formation was assayed after 2 weeks under G418 selection (Y-axis) and used as a measure of toxicity as previously described [ 26 , 43 ]

    Journal: Mobile DNA

    Article Title: The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

    doi: 10.1186/s13100-016-0064-x

    Figure Lengend Snippet: Analysis of select putative phosphorylation sites outside of the endonuclease domain. a Alu retrotransposition: ORF2 proteins containing mutations in the indicated putative phosphorylation sites were used to drive Alu retrotransposition in HeLa cells, as previously described [ 3 ]. ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by Neo R colonies (Y-axis). Asterisks indicate a statistically significant difference in Alu retrotransposition compared to ORF2 ( t -test, P ≤ 0.05). b Acute toxicity: HeLa cells were cotransfected with a Neo R expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. c Acute toxicity: 293 cells were cotransfected with a Neo R expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. In both panels b and c , ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the Neo R expression vector. Colony formation was assayed after 2 weeks under G418 selection (Y-axis) and used as a measure of toxicity as previously described [ 26 , 43 ]

    Article Snippet: ORF2 putative phosphorylation mutants Mutations were introduced into a previously reported [ ] codon-optimized ORF2 expression plasmid (pBudCE4.1, Invitrogen) using the QuikChange Site-Directed Mutagenesis kit (Stratagene) per the manufacturer’s protocol.

    Techniques: Functional Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Selection

    Expression of EN proteins containing mutations in putative phosphorylation sites can induce DNA damage. a Representative western blot analysis of total cell lysates harvested from HeLa cells transiently transfected with the indicated EN putative phosphorylation mutant plasmids. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control lanes indicate cells transfected with an empty vector. a Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 endonuclease domain [ 42 , 43 ], top panel; anti-γH2AX antibodies to detect the phosphorylation of histone H2AX in response to DNA damage, middle panel; and anti-GAPDH to serve as a loading control, bottom panel. b Western blot quantitation. For each sample, the signal detected for ENp was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp. Asterisk denotes a significant difference in the steady-state levels relative to the functional ENp ( t -test, P ≤ 0.05). c Western blot quantitation. For each sample, the signal detected for γH2AX was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp

    Journal: Mobile DNA

    Article Title: The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

    doi: 10.1186/s13100-016-0064-x

    Figure Lengend Snippet: Expression of EN proteins containing mutations in putative phosphorylation sites can induce DNA damage. a Representative western blot analysis of total cell lysates harvested from HeLa cells transiently transfected with the indicated EN putative phosphorylation mutant plasmids. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control lanes indicate cells transfected with an empty vector. a Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 endonuclease domain [ 42 , 43 ], top panel; anti-γH2AX antibodies to detect the phosphorylation of histone H2AX in response to DNA damage, middle panel; and anti-GAPDH to serve as a loading control, bottom panel. b Western blot quantitation. For each sample, the signal detected for ENp was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp. Asterisk denotes a significant difference in the steady-state levels relative to the functional ENp ( t -test, P ≤ 0.05). c Western blot quantitation. For each sample, the signal detected for γH2AX was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp

    Article Snippet: ORF2 putative phosphorylation mutants Mutations were introduced into a previously reported [ ] codon-optimized ORF2 expression plasmid (pBudCE4.1, Invitrogen) using the QuikChange Site-Directed Mutagenesis kit (Stratagene) per the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Transfection, Mutagenesis, Functional Assay, Plasmid Preparation, Generated, Quantitation Assay

    Mutations in the ORF2 endonuclease domain from full-length L1 loci in the human genome. Bioinformatic analysis using L1Base [ 36 ] revealed numerous mutations in the ORF2 endonuclease domains of 134 intact, full-length L1 loci. Positions of mutations relative to the sequence of the L1.3 ORF2 endonuclease domain are indicated by a blue square above the amino acid residue

    Journal: Mobile DNA

    Article Title: The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

    doi: 10.1186/s13100-016-0064-x

    Figure Lengend Snippet: Mutations in the ORF2 endonuclease domain from full-length L1 loci in the human genome. Bioinformatic analysis using L1Base [ 36 ] revealed numerous mutations in the ORF2 endonuclease domains of 134 intact, full-length L1 loci. Positions of mutations relative to the sequence of the L1.3 ORF2 endonuclease domain are indicated by a blue square above the amino acid residue

    Article Snippet: ORF2 putative phosphorylation mutants Mutations were introduced into a previously reported [ ] codon-optimized ORF2 expression plasmid (pBudCE4.1, Invitrogen) using the QuikChange Site-Directed Mutagenesis kit (Stratagene) per the manufacturer’s protocol.

    Techniques: Sequencing

    Schematic representation of the putative phosphorylation sites within ORF2 mutated in this study. Numbered arrows indicate the locations of the putative phosphorylation sites mutated in this study. The boundaries of the ORF2 endonuclease domain are indicated with green brackets. Plasmids encoding for the full-length ORF2p were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; S29A/S37A/S188A/T189A; S312A; S335A; and S312A/S335A. ORF2 11m contains the following mutations ( red ovals ): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A/S312A/S335A. Plasmids encoding for the endonuclease domain (ENp) alone were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; and S29A/S37A/S188A/T189A. EN 9m contains the following mutations ( blue boxes ): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A. Plasmids encoding for the full-length L1 containing the following mutations within ORF2 were generated: S29A; S33A; S312A; S335A; and S312A/S335A

    Journal: Mobile DNA

    Article Title: The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

    doi: 10.1186/s13100-016-0064-x

    Figure Lengend Snippet: Schematic representation of the putative phosphorylation sites within ORF2 mutated in this study. Numbered arrows indicate the locations of the putative phosphorylation sites mutated in this study. The boundaries of the ORF2 endonuclease domain are indicated with green brackets. Plasmids encoding for the full-length ORF2p were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; S29A/S37A/S188A/T189A; S312A; S335A; and S312A/S335A. ORF2 11m contains the following mutations ( red ovals ): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A/S312A/S335A. Plasmids encoding for the endonuclease domain (ENp) alone were generated with the following mutations: S29A; S33A; S37A; S79A; S188A; S228A; S29A/S37A; S79A/T82A; S188A/T189A; S29A/S37A/S228A; and S29A/S37A/S188A/T189A. EN 9m contains the following mutations ( blue boxes ): S29A/S33A/S37A/S151A/S188A/T189A/T220A/T224A/S228A. Plasmids encoding for the full-length L1 containing the following mutations within ORF2 were generated: S29A; S33A; S312A; S335A; and S312A/S335A

    Article Snippet: ORF2 putative phosphorylation mutants Mutations were introduced into a previously reported [ ] codon-optimized ORF2 expression plasmid (pBudCE4.1, Invitrogen) using the QuikChange Site-Directed Mutagenesis kit (Stratagene) per the manufacturer’s protocol.

    Techniques: Generated

    Acute toxicity assay in HeLa and 293 cells transiently transfected with ORF2 putative phosphorylation mutant plasmids. a HeLa cells were cotransfected with a Neo R expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. b 293 cells were cotransfected with a Neo R expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. In both panel a and b ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the Neo R expression vector. Colony formation was assayed after 2 weeks under G418 selection (Y-axis) and used as a measure of toxicity as previously described [ 26 , 42 ]

    Journal: Mobile DNA

    Article Title: The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

    doi: 10.1186/s13100-016-0064-x

    Figure Lengend Snippet: Acute toxicity assay in HeLa and 293 cells transiently transfected with ORF2 putative phosphorylation mutant plasmids. a HeLa cells were cotransfected with a Neo R expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. b 293 cells were cotransfected with a Neo R expression vector and the indicated ORF2 putative phosphorylation mutant plasmid. In both panel a and b ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the Neo R expression vector. Colony formation was assayed after 2 weeks under G418 selection (Y-axis) and used as a measure of toxicity as previously described [ 26 , 42 ]

    Article Snippet: ORF2 putative phosphorylation mutants Mutations were introduced into a previously reported [ ] codon-optimized ORF2 expression plasmid (pBudCE4.1, Invitrogen) using the QuikChange Site-Directed Mutagenesis kit (Stratagene) per the manufacturer’s protocol.

    Techniques: Transfection, Mutagenesis, Expressing, Plasmid Preparation, Functional Assay, Selection

    Alu retrotransposition driven by ORF2 proteins containing mutations in putative phosphorylation sites. ORF2 proteins containing mutations in the indicated putative phosphorylation sites were used to drive Alu retrotransposition in HeLa cells, as previously described [ 3 ]. ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by Neo R colonies (Y-axis). Asterisks indicate a statistically significant difference in Alu retrotransposition compared to ORF2 ( t -test, P ≤ 0.05)

    Journal: Mobile DNA

    Article Title: The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

    doi: 10.1186/s13100-016-0064-x

    Figure Lengend Snippet: Alu retrotransposition driven by ORF2 proteins containing mutations in putative phosphorylation sites. ORF2 proteins containing mutations in the indicated putative phosphorylation sites were used to drive Alu retrotransposition in HeLa cells, as previously described [ 3 ]. ORF2 is the functional protein and ORF2 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control indicates cells transfected with an empty vector and the Alu retrotransposition reporter plasmid. The graph depicts the relative number of Alu retrotransposition events as represented by Neo R colonies (Y-axis). Asterisks indicate a statistically significant difference in Alu retrotransposition compared to ORF2 ( t -test, P ≤ 0.05)

    Article Snippet: ORF2 putative phosphorylation mutants Mutations were introduced into a previously reported [ ] codon-optimized ORF2 expression plasmid (pBudCE4.1, Invitrogen) using the QuikChange Site-Directed Mutagenesis kit (Stratagene) per the manufacturer’s protocol.

    Techniques: Functional Assay, Transfection, Plasmid Preparation