gluc  (New England Biolabs)


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  • 96
    Name:
    Anti GLuc Antibody
    Description:
    Anti GLuc Antibody 200 ul
    Catalog Number:
    e8023s
    Price:
    246
    Size:
    200 ul
    Category:
    Cellular Biology
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    Structured Review

    New England Biolabs gluc
    Anti GLuc Antibody
    Anti GLuc Antibody 200 ul
    https://www.bioz.com/result/gluc/product/New England Biolabs
    Average 96 stars, based on 97 article reviews
    Price from $9.99 to $1999.99
    gluc - by Bioz Stars, 2020-08
    96/100 stars

    Images

    1) Product Images from "Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines"

    Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

    Journal: Viruses

    doi: 10.3390/v11100928

    Contribution of PB2 and PB1 mutations of PR8/AA and PR8/Len in the viral polymerase activity at different temperatures. HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of the indicated combinations of ambisense pDZ expression plasmids encoding the PB2 and PB1 from PR8/WT, PR8/AA and PR8/Len together with the pDZ encoding PA and NP PR8/WT proteins; together with 500 ng of the hPol-I-GFP ( A ) and -Gluc ( B ), and 100 ng of SV40 Cluc to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and reporter gene expression was evaluated 24 h later by GFP imaging ( A ) and Gluc expression ( B ). Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that in the absence of pDZ NP plasmid. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P
    Figure Legend Snippet: Contribution of PB2 and PB1 mutations of PR8/AA and PR8/Len in the viral polymerase activity at different temperatures. HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of the indicated combinations of ambisense pDZ expression plasmids encoding the PB2 and PB1 from PR8/WT, PR8/AA and PR8/Len together with the pDZ encoding PA and NP PR8/WT proteins; together with 500 ng of the hPol-I-GFP ( A ) and -Gluc ( B ), and 100 ng of SV40 Cluc to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and reporter gene expression was evaluated 24 h later by GFP imaging ( A ) and Gluc expression ( B ). Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that in the absence of pDZ NP plasmid. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P

    Techniques Used: Activity Assay, Transfection, Expressing, Imaging, Plasmid Preparation

    Effect of temperature on the polymerase activity of PR8/AA and PR8/Len. ( A ) Schematic representation of segments 1 (PB2), 2 (PB1) and 8 (NS) of PR8/WT (white, left), PR8/AA (black, middle) and PR8/Len (grey, right). Amino acid substitutions to generate PR8/AA and PR8/Len are indicated. B-C) Minigenome activity: HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of ambisense pDZ expression plasmids encoding the minimal requirements for viral genome replication and gene transcription (PB2, PB1, PA, and NP) together with 500 ng of a vRNA-like expression plasmid encoding GFP ( B ) or Gluc ( C ) under the control of the human polymerase I promoter (hPol-I-GFP and -Gluc, respectively), and 100 ng of an SV40 Cluc expressing plasmid to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and viral replication and transcription was evaluated 24 h later by GFP ( B ) and luciferase ( C ) expression. Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that obtained in the absence of pDZ NP. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P
    Figure Legend Snippet: Effect of temperature on the polymerase activity of PR8/AA and PR8/Len. ( A ) Schematic representation of segments 1 (PB2), 2 (PB1) and 8 (NS) of PR8/WT (white, left), PR8/AA (black, middle) and PR8/Len (grey, right). Amino acid substitutions to generate PR8/AA and PR8/Len are indicated. B-C) Minigenome activity: HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of ambisense pDZ expression plasmids encoding the minimal requirements for viral genome replication and gene transcription (PB2, PB1, PA, and NP) together with 500 ng of a vRNA-like expression plasmid encoding GFP ( B ) or Gluc ( C ) under the control of the human polymerase I promoter (hPol-I-GFP and -Gluc, respectively), and 100 ng of an SV40 Cluc expressing plasmid to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and viral replication and transcription was evaluated 24 h later by GFP ( B ) and luciferase ( C ) expression. Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that obtained in the absence of pDZ NP. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P

    Techniques Used: Activity Assay, Transfection, Expressing, Plasmid Preparation, Luciferase

    2) Product Images from "The Functionality of Minimal PiggyBac Transposons in Mammalian Cells"

    Article Title: The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2016.76

    Schematic of minimal piggyBac mPB-GLuc-mCherry ( a ) and nontransposon GLuc-mCherry ( b ) bicistronic vectors. Both plasmids contained secretable Gaussia luciferase ( GLuc ) and mCherry genes separated with an internal ribosomal entry site (IRES) for synchronized expression from a cytomegalovirus (CMV) promoter and a bGH polyadenylation signal ( pA ) termination. Minimal terminal repeats ( 5′TRmin, 3′TRmin ) and the piggyBac transposase open reading frame (ORF) ( PBase ) with truncated terminal domains ( 5′TD ( trunc. ) , 3′TD ( trunc. )) was present only in mPB-GLuc-mCherry vector. PiggyBac transposase expression was controlled by the phosphoglycerate kinase (PGK) promoter as described for other minimal piggyBac plasmids. ( c ) Expression of mCherry in mice tails 3 days, 30 days, and 6 months after local injection/electroporation (arrows) of GLuc-mCherry and mPB-GLuc-mCherry vectors. Control mouse represents the background signal in a nontransfected mouse. ( d ) Indirect enzyme-linked immunosorbent assay (ELISA) showing production of antibodies against GLuc in mouse serum after a single delivery of either a nontransposon vector GLuc-mCherry or a minimal piggyBac vector mPB-GLuc-mCherry .
    Figure Legend Snippet: Schematic of minimal piggyBac mPB-GLuc-mCherry ( a ) and nontransposon GLuc-mCherry ( b ) bicistronic vectors. Both plasmids contained secretable Gaussia luciferase ( GLuc ) and mCherry genes separated with an internal ribosomal entry site (IRES) for synchronized expression from a cytomegalovirus (CMV) promoter and a bGH polyadenylation signal ( pA ) termination. Minimal terminal repeats ( 5′TRmin, 3′TRmin ) and the piggyBac transposase open reading frame (ORF) ( PBase ) with truncated terminal domains ( 5′TD ( trunc. ) , 3′TD ( trunc. )) was present only in mPB-GLuc-mCherry vector. PiggyBac transposase expression was controlled by the phosphoglycerate kinase (PGK) promoter as described for other minimal piggyBac plasmids. ( c ) Expression of mCherry in mice tails 3 days, 30 days, and 6 months after local injection/electroporation (arrows) of GLuc-mCherry and mPB-GLuc-mCherry vectors. Control mouse represents the background signal in a nontransfected mouse. ( d ) Indirect enzyme-linked immunosorbent assay (ELISA) showing production of antibodies against GLuc in mouse serum after a single delivery of either a nontransposon vector GLuc-mCherry or a minimal piggyBac vector mPB-GLuc-mCherry .

    Techniques Used: Luciferase, Expressing, Plasmid Preparation, Mouse Assay, Injection, Electroporation, Indirect ELISA, Enzyme-linked Immunosorbent Assay

    3) Product Images from "A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization"

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32581-1

    Compound screen identifies small molecules inhibiting PrP dimerization and conversion. ( a ) The Pharmacologically Active Compound Library (Selleckchem) was screened in RK13-DC cells for compounds reducing bioluminescence at a concentration of 10 µM after a 24-hour treatment. From among the 1,650 compounds 240 were found to reduce bioluminescence between 20–85%. From among these 73 compounds were found to reduce bioluminescence in a counter screen in RK13 cells stably expressing full-length Gaussia luciferase (RK13-GLuc cells), suggesting that they were either toxic or negatively affecting luciferase activity. From among the remaining 167 compounds, 13 among the most potent to reduce dimerization without negatively affecting bioluminescence of RK13-GLuc cells were selected for further analysis of their anti-prion activity in ScN2a cells. ( b ) The 13 selected compounds strongly reduced bioluminescence of RK13-DC cells by 60–80%. ( c ) From among these 13 compounds the most potent to reduce bioluminescence was JTC-801, a quinoline derivative. ( d ) The EC 50 for the reduction of bioluminescence of RK13-DC cells for JTC-801 was close to 2 µM, a concentration that did not affect cell survival within 24 h. ( e , f ) Biochemical and densitometric analysis shows that a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected ScN2a cells with an EC 50 of 370 nM. For presentation purposes additional lanes were excised from the image. Treatment of ScN2a cells with JTC-801 for 5 days did not affect cell for survival for concentrations ≤1.0 µM. ( g , h ) Similarly, a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected SMB cells with an EC 50 of 220 nM without being toxic for concentrations up to 1 µM. In ( b , d , f , h ) error bars indicate SD.
    Figure Legend Snippet: Compound screen identifies small molecules inhibiting PrP dimerization and conversion. ( a ) The Pharmacologically Active Compound Library (Selleckchem) was screened in RK13-DC cells for compounds reducing bioluminescence at a concentration of 10 µM after a 24-hour treatment. From among the 1,650 compounds 240 were found to reduce bioluminescence between 20–85%. From among these 73 compounds were found to reduce bioluminescence in a counter screen in RK13 cells stably expressing full-length Gaussia luciferase (RK13-GLuc cells), suggesting that they were either toxic or negatively affecting luciferase activity. From among the remaining 167 compounds, 13 among the most potent to reduce dimerization without negatively affecting bioluminescence of RK13-GLuc cells were selected for further analysis of their anti-prion activity in ScN2a cells. ( b ) The 13 selected compounds strongly reduced bioluminescence of RK13-DC cells by 60–80%. ( c ) From among these 13 compounds the most potent to reduce bioluminescence was JTC-801, a quinoline derivative. ( d ) The EC 50 for the reduction of bioluminescence of RK13-DC cells for JTC-801 was close to 2 µM, a concentration that did not affect cell survival within 24 h. ( e , f ) Biochemical and densitometric analysis shows that a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected ScN2a cells with an EC 50 of 370 nM. For presentation purposes additional lanes were excised from the image. Treatment of ScN2a cells with JTC-801 for 5 days did not affect cell for survival for concentrations ≤1.0 µM. ( g , h ) Similarly, a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected SMB cells with an EC 50 of 220 nM without being toxic for concentrations up to 1 µM. In ( b , d , f , h ) error bars indicate SD.

    Techniques Used: Concentration Assay, Stable Transfection, Expressing, Luciferase, Activity Assay, Infection

    4) Product Images from "High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production"

    Article Title: High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00378

    High-throughput screening strategy and results. (A) Schematic of screen design. Addition of 1 μg/mL doxycycline (Dox) to the stable single-colony Saos-2 GLuc.Col cell line induces expression of the eGLuc2.Colα2(I) fusion protein, which can be proteolytically processed post-secretion. Luminescence generated by the fusion protein may be assayed upon addition of the eGLuc2 substrate. (B) eGLuc2.Colα2(I) from Saos-2 GLuc.Col lysate was immunoprecipitated with an α-GLuc antibody and analyzed by immunoblotting, revealing that eGLuc2.Colα2(I) associates intracellularly with Colα1(I). (C) Disulfide-dependent assembly of eGLuc2.Colα2(I) with Colα1(I). Collagen-I precipitated from conditioned media of Saos-2 GLuc.Col cells was treated with iodoacetamide and heated in Laemmli buffer with or without DTT. Samples were separated by SDS-PAGE and analyzed by immunoblotting. (D) Plot of filtered screening results. (E) Validated small molecule modulators of collagen-I secretion.
    Figure Legend Snippet: High-throughput screening strategy and results. (A) Schematic of screen design. Addition of 1 μg/mL doxycycline (Dox) to the stable single-colony Saos-2 GLuc.Col cell line induces expression of the eGLuc2.Colα2(I) fusion protein, which can be proteolytically processed post-secretion. Luminescence generated by the fusion protein may be assayed upon addition of the eGLuc2 substrate. (B) eGLuc2.Colα2(I) from Saos-2 GLuc.Col lysate was immunoprecipitated with an α-GLuc antibody and analyzed by immunoblotting, revealing that eGLuc2.Colα2(I) associates intracellularly with Colα1(I). (C) Disulfide-dependent assembly of eGLuc2.Colα2(I) with Colα1(I). Collagen-I precipitated from conditioned media of Saos-2 GLuc.Col cells was treated with iodoacetamide and heated in Laemmli buffer with or without DTT. Samples were separated by SDS-PAGE and analyzed by immunoblotting. (D) Plot of filtered screening results. (E) Validated small molecule modulators of collagen-I secretion.

    Techniques Used: High Throughput Screening Assay, Expressing, Generated, Immunoprecipitation, SDS Page

    5) Product Images from "Easy detection of hormone secretion from LβT2 cells by using Gaussia luciferase"

    Article Title: Easy detection of hormone secretion from LβT2 cells by using Gaussia luciferase

    Journal: The Journal of Reproduction and Development

    doi: 10.1262/jrd.2016-174

    Time-dependent increment of Gluc activity in the LβT2-cultured medium (A) and NIH3T3-cultured medium (B), and Gluc protein secretion in LβT2-cultured medium (C). The cells were transfected with pCMV-Gluc2. (A and B) Gluc-expressing cells were incubated for the indicated times without treatment (open circle), and in the presence of 10 nM GnRH (closed circle), or 50 mM KCl (open triangle). Gluc activity in the medium at each time point was measured as described in “Materials and Methods”. The asterisks indicate that the Gluc activities are significantly different from that in the absence of agents, * P
    Figure Legend Snippet: Time-dependent increment of Gluc activity in the LβT2-cultured medium (A) and NIH3T3-cultured medium (B), and Gluc protein secretion in LβT2-cultured medium (C). The cells were transfected with pCMV-Gluc2. (A and B) Gluc-expressing cells were incubated for the indicated times without treatment (open circle), and in the presence of 10 nM GnRH (closed circle), or 50 mM KCl (open triangle). Gluc activity in the medium at each time point was measured as described in “Materials and Methods”. The asterisks indicate that the Gluc activities are significantly different from that in the absence of agents, * P

    Techniques Used: Activity Assay, Cell Culture, Transfection, Expressing, Incubation

    Effect of antide on GnRH-induced Gluc activity (A), on GnRH-induced LH secretion (B), and the difference in Gluc activity in the absence and presence of antide (C) in LβT2 cells. (A) LβT2 cells were transfected with pCMV-Gluc2. Gluc-expressing cells were stimulated with 10 nM GnRH or 50 mM KCl for 2 h in the presence (closed column) or absence (open column) of 100 nM antide. Results show the relative value, where the relative light unit of the vehicle without antide is expressed as 1. Asterisks indicate that the Gluc activities are significantly different from those in the absence of antide. ** P
    Figure Legend Snippet: Effect of antide on GnRH-induced Gluc activity (A), on GnRH-induced LH secretion (B), and the difference in Gluc activity in the absence and presence of antide (C) in LβT2 cells. (A) LβT2 cells were transfected with pCMV-Gluc2. Gluc-expressing cells were stimulated with 10 nM GnRH or 50 mM KCl for 2 h in the presence (closed column) or absence (open column) of 100 nM antide. Results show the relative value, where the relative light unit of the vehicle without antide is expressed as 1. Asterisks indicate that the Gluc activities are significantly different from those in the absence of antide. ** P

    Techniques Used: Activity Assay, Transfection, Expressing

    Secretagogue-induced Gluc activity in AtT20-cultured medium (A) and ACTH secretion from AtT20 cells (B); secretagogue-induced Gluc activity in GnRH receptor-expressing HEK293-cultured medium (C), and GnRH-induced SRE activity of GnRH receptor-expressing HEK293 cells (D). (A) AtT20 cells were transfected with pCMV-Gluc2. Gluc-expressing cells were stimulated with 10 nM CRH or a vehicle for 4 h. Results are indicated as shown in Fig. 2 . The relative light unit of the vehicle is expressed as 1. (B) AtT20 cells were stimulated with 10 nM CRH for 4 h. Results show the amount of ACTH secreted in the medium as the percentage of the total amount of ACTH contained in the medium and in the cells. Asterisks indicate that the secreted ACTH amount is significant. ** P
    Figure Legend Snippet: Secretagogue-induced Gluc activity in AtT20-cultured medium (A) and ACTH secretion from AtT20 cells (B); secretagogue-induced Gluc activity in GnRH receptor-expressing HEK293-cultured medium (C), and GnRH-induced SRE activity of GnRH receptor-expressing HEK293 cells (D). (A) AtT20 cells were transfected with pCMV-Gluc2. Gluc-expressing cells were stimulated with 10 nM CRH or a vehicle for 4 h. Results are indicated as shown in Fig. 2 . The relative light unit of the vehicle is expressed as 1. (B) AtT20 cells were stimulated with 10 nM CRH for 4 h. Results show the amount of ACTH secreted in the medium as the percentage of the total amount of ACTH contained in the medium and in the cells. Asterisks indicate that the secreted ACTH amount is significant. ** P

    Techniques Used: Activity Assay, Cell Culture, Expressing, Transfection

    6) Product Images from "Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice"

    Article Title: Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice

    Journal: Journal of Extracellular Vesicles

    doi: 10.3402/jev.v4.26238

    Stability of exosome labelling by gLuc-LA in serum. (a) Time-course of stability of gLuc activity of gLuc-LA-labelled B16BL6 exosomes at 37°C in 20% FBS/PBS buffer. The initial gLuc activity was about 10 6 RLU/s/10 µL. (b) Time-course of exosome labelling stability of gLuc-LA at 37°C in 20% FBS/PBS buffer of 4 samples.
    Figure Legend Snippet: Stability of exosome labelling by gLuc-LA in serum. (a) Time-course of stability of gLuc activity of gLuc-LA-labelled B16BL6 exosomes at 37°C in 20% FBS/PBS buffer. The initial gLuc activity was about 10 6 RLU/s/10 µL. (b) Time-course of exosome labelling stability of gLuc-LA at 37°C in 20% FBS/PBS buffer of 4 samples.

    Techniques Used: Activity Assay

    7) Product Images from "RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii"

    Article Title: RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1523230113

    Characterization of the DUS16 mutant. CC-124, wild-type strain; Gluc(1×), CC-124 transgenic strain expressing Gaussia luciferase ( gluc ) bearing a sequence perfectly complementary to Chlamydomonas miR9897-3p; dus16-1 , DUS16 -defective mutant of
    Figure Legend Snippet: Characterization of the DUS16 mutant. CC-124, wild-type strain; Gluc(1×), CC-124 transgenic strain expressing Gaussia luciferase ( gluc ) bearing a sequence perfectly complementary to Chlamydomonas miR9897-3p; dus16-1 , DUS16 -defective mutant of

    Techniques Used: Mutagenesis, Transgenic Assay, Expressing, Luciferase, Sequencing

    8) Product Images from "Arenavirus reverse genetics for vaccine development"

    Article Title: Arenavirus reverse genetics for vaccine development

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.051102-0

    A two-plasmid arenavirus rescue system. (a) Schematic representation of the two-plasmid system for the generation of rLCMV and rCandid#1: Plasmids encoding either NP and the vRNA L segment (pC-NP/hpPol-I L), or the L protein and the vRNA S segment (pC-L/hpPol-I S) for the generation of recombinant arenavirus using the two-plasmid approach are indicated. (b, c) Comparative activity of pC and pC/hpPol-I-based expression plasmids in a MG assay. Human 293T cells were co-transfected with the LCMV dual-reporter MG plasmid and the pC expression plasmids encoding the viral NP and L (four-plasmid viral rescue) or the pC/hpPol-I (two-plasmid viral rescue) expression plasmids for LCMV, together with pSV40-Cluc vector to normalize transfection efficiencies. As a negative control, cells were co-transfected only with pC-L or pC-L/hpPol-I S (-NP), using empty pC to keep constant the total amount of transfected DNA. At 48 h p.t., MG expression was analysed by GFP using fluorescence microscopy (b) and TCSs were collected and analysed for Gluc and Cluc expression (c). Representative images of three independent experiments and fold induction over the negative controls are represented. Scale bar, 100 µm.
    Figure Legend Snippet: A two-plasmid arenavirus rescue system. (a) Schematic representation of the two-plasmid system for the generation of rLCMV and rCandid#1: Plasmids encoding either NP and the vRNA L segment (pC-NP/hpPol-I L), or the L protein and the vRNA S segment (pC-L/hpPol-I S) for the generation of recombinant arenavirus using the two-plasmid approach are indicated. (b, c) Comparative activity of pC and pC/hpPol-I-based expression plasmids in a MG assay. Human 293T cells were co-transfected with the LCMV dual-reporter MG plasmid and the pC expression plasmids encoding the viral NP and L (four-plasmid viral rescue) or the pC/hpPol-I (two-plasmid viral rescue) expression plasmids for LCMV, together with pSV40-Cluc vector to normalize transfection efficiencies. As a negative control, cells were co-transfected only with pC-L or pC-L/hpPol-I S (-NP), using empty pC to keep constant the total amount of transfected DNA. At 48 h p.t., MG expression was analysed by GFP using fluorescence microscopy (b) and TCSs were collected and analysed for Gluc and Cluc expression (c). Representative images of three independent experiments and fold induction over the negative controls are represented. Scale bar, 100 µm.

    Techniques Used: Plasmid Preparation, Recombinant, Activity Assay, Expressing, Transfection, Negative Control, Fluorescence, Microscopy

    The polymerase I terminator (Pol-I T) is more efficient than the hepatitis delta virus ribozyme (HDVR) in promoting RNA replication and transcription of a LCMV S genome analogue. (a) Schematic representation of the reporter gene expression plasmids. Dual-reporter LCMV S RNA analogue plasmids encoding Pur-GFP and Gluc instead of the viral NP and GP, respectively, were inserted between the human RNA polymerase I promoter (hPol-I) and terminator (T, top) or the HDVR (bottom) sequences. Viral untranslated regions (UTR) and intergenic region (IGR) are indicated. (b, c) Comparison of the Pol-I T and HDVR sequences. Human 293T cells were co-transfected with the hpPol-I Gluc/Pur-GFP HDVR or hpPol-I Gluc/Pur-GFP T dual-reporter plasmids together with the pC expression plasmids for LCMV NP and L, together with pSV40-Cluc to normalize transfection efficiencies. As negative control cells were transfected without pC-NP, using empty pC to keep the total amount of transfected DNA constant. At 48 h p.t., GFP-expressing cells were detected by fluorescence microscopy (b). TCSs from the same transfections were analysed for levels of Gluc and Cluc activities (c); representative GFP expression images and Gluc fold induction over the negative controls for three independent experiments are shown. Scale bar, 100 µm.
    Figure Legend Snippet: The polymerase I terminator (Pol-I T) is more efficient than the hepatitis delta virus ribozyme (HDVR) in promoting RNA replication and transcription of a LCMV S genome analogue. (a) Schematic representation of the reporter gene expression plasmids. Dual-reporter LCMV S RNA analogue plasmids encoding Pur-GFP and Gluc instead of the viral NP and GP, respectively, were inserted between the human RNA polymerase I promoter (hPol-I) and terminator (T, top) or the HDVR (bottom) sequences. Viral untranslated regions (UTR) and intergenic region (IGR) are indicated. (b, c) Comparison of the Pol-I T and HDVR sequences. Human 293T cells were co-transfected with the hpPol-I Gluc/Pur-GFP HDVR or hpPol-I Gluc/Pur-GFP T dual-reporter plasmids together with the pC expression plasmids for LCMV NP and L, together with pSV40-Cluc to normalize transfection efficiencies. As negative control cells were transfected without pC-NP, using empty pC to keep the total amount of transfected DNA constant. At 48 h p.t., GFP-expressing cells were detected by fluorescence microscopy (b). TCSs from the same transfections were analysed for levels of Gluc and Cluc activities (c); representative GFP expression images and Gluc fold induction over the negative controls for three independent experiments are shown. Scale bar, 100 µm.

    Techniques Used: Expressing, Transfection, Negative Control, Fluorescence, Microscopy

    Specificity of the Pol-I promoter. (a) Schematic representation of plasmids expressing dual-reporter LCMV S RNA analogues (MG) under the hPol-I (top) or mPol-I (bottom) promoters: dual-reporter LCMV S RNA analogue plasmids encoding Pur-GFP and Gluc instead of the viral NP and GP, were inserted between the human RNA polymerase I promoter (hPol-I, top) or the murine RNA polymerase I promoter (mPol-I, bottom) and the polymerase terminator (Pol-I T) sequences. (b, c) Species specificity of the Pol-I promoter. Human 293T cells and rodent BHK-21 cells were co-transfected with the pC LCMV NP and L expression plasmids together with hPol-I or mPol-I Gluc/Pur-GFP reporter plasmids, together with pSV40-Cluc vector to normalize transfection efficiencies. As negative control, cells were co-transfected only with LCMV pC-L, using empty pC to keep constant the total amount of transfected DNA. At 48 h p.t., GFP expression was detected by fluorescence microscopy (b) and TCSs were analysed for the presence of secreted Gluc and Cluc (c); representative GFP expression images and Gluc fold induction over the negative controls for three independent experiments are shown. Scale bar, 100 µm.
    Figure Legend Snippet: Specificity of the Pol-I promoter. (a) Schematic representation of plasmids expressing dual-reporter LCMV S RNA analogues (MG) under the hPol-I (top) or mPol-I (bottom) promoters: dual-reporter LCMV S RNA analogue plasmids encoding Pur-GFP and Gluc instead of the viral NP and GP, were inserted between the human RNA polymerase I promoter (hPol-I, top) or the murine RNA polymerase I promoter (mPol-I, bottom) and the polymerase terminator (Pol-I T) sequences. (b, c) Species specificity of the Pol-I promoter. Human 293T cells and rodent BHK-21 cells were co-transfected with the pC LCMV NP and L expression plasmids together with hPol-I or mPol-I Gluc/Pur-GFP reporter plasmids, together with pSV40-Cluc vector to normalize transfection efficiencies. As negative control, cells were co-transfected only with LCMV pC-L, using empty pC to keep constant the total amount of transfected DNA. At 48 h p.t., GFP expression was detected by fluorescence microscopy (b) and TCSs were analysed for the presence of secreted Gluc and Cluc (c); representative GFP expression images and Gluc fold induction over the negative controls for three independent experiments are shown. Scale bar, 100 µm.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Negative Control, Fluorescence, Microscopy

    Rescue of recombinant tri-segmented (r3) LCMV and Candid#1 in Vero cells. (a) Schematic representation of the plasmid used for the generation of r3LCMV and r3Candid#1. (b, c) Virus rescue. Protein expression plasmids (pC) encoding NP and L were co-transfected together with the respective LCMV (b) or Candid#1 (c) hpPol-I GP/GFP, hpPol-I Gluc/NP and hpPol-I L plasmids into Vero cells. At 72 h p.t., cells were transferred into 10 cm dishes for an additional 72 h before TCSs were collected and assessed for virus rescue based on their infectivity in fresh Vero cells by GFP expression [b(i) and c(i)]. Representative images of at least three independent virus rescues are shown. Scale bar, 100 µm. TCSs from infected Vero cells were collected to assess levels of Gluc activity [b(ii) and c(ii)]. Mock-infected cells were included as negative controls. Cell lysates were prepared and analysed for protein expression by WB [b(iii) and c(iii)] using a monoclonal antibody for GFP or a polyclonal antibody for Gluc. LCMV and Candid#1 NP expressions were assessed with monoclonal antibodies. GAPDH protein expression was included as a loading control.
    Figure Legend Snippet: Rescue of recombinant tri-segmented (r3) LCMV and Candid#1 in Vero cells. (a) Schematic representation of the plasmid used for the generation of r3LCMV and r3Candid#1. (b, c) Virus rescue. Protein expression plasmids (pC) encoding NP and L were co-transfected together with the respective LCMV (b) or Candid#1 (c) hpPol-I GP/GFP, hpPol-I Gluc/NP and hpPol-I L plasmids into Vero cells. At 72 h p.t., cells were transferred into 10 cm dishes for an additional 72 h before TCSs were collected and assessed for virus rescue based on their infectivity in fresh Vero cells by GFP expression [b(i) and c(i)]. Representative images of at least three independent virus rescues are shown. Scale bar, 100 µm. TCSs from infected Vero cells were collected to assess levels of Gluc activity [b(ii) and c(ii)]. Mock-infected cells were included as negative controls. Cell lysates were prepared and analysed for protein expression by WB [b(iii) and c(iii)] using a monoclonal antibody for GFP or a polyclonal antibody for Gluc. LCMV and Candid#1 NP expressions were assessed with monoclonal antibodies. GAPDH protein expression was included as a loading control.

    Techniques Used: Recombinant, Plasmid Preparation, Expressing, Transfection, Infection, Activity Assay, Western Blot

    9) Product Images from "SERCaMP: a carboxy-terminal protein modification that enables monitoring of ER calcium homeostasis"

    Article Title: SERCaMP: a carboxy-terminal protein modification that enables monitoring of ER calcium homeostasis

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-06-1141

    AAV-GLuc-ASARTDL is responsive to thapsigargin in an in vivo model. (A) Schema of the in vivo SERCaMP assay. Rats were intrahepatically injected with AAV-GLuc, blood was collected from tail, and plasma was assayed for GLuc activity. (B) Transgene expression in liver was examined by immunohistochemistry. Rats were intrahepatically injected with GLuc-ASARTDL or AAV-GFP and stained with anti-GLuc (red) to examine expression. The viral titer injected (3 × 10 11 vg) was ∼400-fold higher than in E (7.6 × 10 8 vg) to allow for detection by immunohistochemistry. Nuclei were stained with DAPI (blue). Scale bar, 200 μm. (C) Half-life of GLuc proteins in vivo. Conditioned medium collected from stable SH-SY5Y cells was normalized for enzymatic activity and injected into WT rats fitted with a back port connected to the jugular vein, and blood was collected at indicated time points. Half-life is estimated to be
    Figure Legend Snippet: AAV-GLuc-ASARTDL is responsive to thapsigargin in an in vivo model. (A) Schema of the in vivo SERCaMP assay. Rats were intrahepatically injected with AAV-GLuc, blood was collected from tail, and plasma was assayed for GLuc activity. (B) Transgene expression in liver was examined by immunohistochemistry. Rats were intrahepatically injected with GLuc-ASARTDL or AAV-GFP and stained with anti-GLuc (red) to examine expression. The viral titer injected (3 × 10 11 vg) was ∼400-fold higher than in E (7.6 × 10 8 vg) to allow for detection by immunohistochemistry. Nuclei were stained with DAPI (blue). Scale bar, 200 μm. (C) Half-life of GLuc proteins in vivo. Conditioned medium collected from stable SH-SY5Y cells was normalized for enzymatic activity and injected into WT rats fitted with a back port connected to the jugular vein, and blood was collected at indicated time points. Half-life is estimated to be

    Techniques Used: In Vivo, Injection, Activity Assay, Expressing, Immunohistochemistry, Staining

    10) Product Images from "RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii"

    Article Title: RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1523230113

    Characterization of the DUS16 mutant. CC-124, wild-type strain; Gluc(1×), CC-124 transgenic strain expressing Gaussia luciferase ( gluc ) bearing a sequence perfectly complementary to Chlamydomonas miR9897-3p; dus16-1 , DUS16 -defective mutant of
    Figure Legend Snippet: Characterization of the DUS16 mutant. CC-124, wild-type strain; Gluc(1×), CC-124 transgenic strain expressing Gaussia luciferase ( gluc ) bearing a sequence perfectly complementary to Chlamydomonas miR9897-3p; dus16-1 , DUS16 -defective mutant of

    Techniques Used: Mutagenesis, Transgenic Assay, Expressing, Luciferase, Sequencing

    11) Product Images from "A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization"

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32581-1

    PrP-NGLuc and PrP-CGLuc expression in RK13-DC cells results in bioluminescence. ( a ) Western blot analysis with the Sha31 antibody against PrP shows that RK13-DC cells stably express PrP-NGLuc and PrP-CGLuc from a bicistronic expression vector (lanes 3 and 4). Non-transfected RK13 cells do not express PrP and do not show a detectable signal for PrP (lanes 1 and 2), whereas N2a cells show the characteristic signal for wild-type PrP (lanes 5 and 6). Deglycosylation of cell lysates with peptide-N-glycosidase F (PNGase F, lanes 2, 4, and 6) resulted in lower molecular weight bands suggesting that mature PrP-NGLuc and PrP-CGLuc are properly glycosylated in RK13-DC cells as PrP is in N2a cells. Detection of tubulin on the same blot served as a loading control. Additional lanes were excised for presentation purposes. ( b ) Western blot analysis with the Sha31 antibody against PrP shows that treatment of RK13-DC cells with increasing amounts of phosphoinositide phospholipase C (PI-PLC) increases the amount of detectable protein released into cell culture medium suggesting that mature PrP-NGLuc and PrP-CGLuc are anchored to the outer cell membrane by GPI. Additional lanes were excised for presentation purposes. ( c ) Densitometric quantification of the PrP signal from three different experiments as shown in ( b ). ( d ) Life cell immunofluorescence staining with the Sha31 antibody to PrP (left panels) shows that PrP-NGLuc and PrP-CGLuc are expressed in RK13-DC cells (top row), similar to wild-type PrP in N2a cells (bottom row). Bright-field microscopy (centre panels) shows the contours of the imaged cells. An overlay of fluorescence and bright-field images (right panels) shows that PrP-NGLuc, PrP-CGLuc, and PrP are located at the cell membrane. Bar = 20 µm. ( e ) The bioluminescence measured from RK13-DC cells expressing both PrP-NGLuc and PrP-CGLuc was more than 10-fold above background levels measured from RK13 cells stably expressing only PrP-NGLuc or PrP-CGLuc, or both NGLuc and CGLuc together each lacking the PrP moiety. A functional luciferase consisting of its N- and C-terminal halves was only formed in RK13-DC cells where dimers between the PrP moieties of PrP-NGLuc and PrP-CGLuc could form. In comparison to PrP-NGLuc and PrP-CGLuc in RK13-DC cells, bioluminescence of full-length GLuc in RK13 cells was almost 17-fold higher. In ( c , e ) error bars indicate SD.
    Figure Legend Snippet: PrP-NGLuc and PrP-CGLuc expression in RK13-DC cells results in bioluminescence. ( a ) Western blot analysis with the Sha31 antibody against PrP shows that RK13-DC cells stably express PrP-NGLuc and PrP-CGLuc from a bicistronic expression vector (lanes 3 and 4). Non-transfected RK13 cells do not express PrP and do not show a detectable signal for PrP (lanes 1 and 2), whereas N2a cells show the characteristic signal for wild-type PrP (lanes 5 and 6). Deglycosylation of cell lysates with peptide-N-glycosidase F (PNGase F, lanes 2, 4, and 6) resulted in lower molecular weight bands suggesting that mature PrP-NGLuc and PrP-CGLuc are properly glycosylated in RK13-DC cells as PrP is in N2a cells. Detection of tubulin on the same blot served as a loading control. Additional lanes were excised for presentation purposes. ( b ) Western blot analysis with the Sha31 antibody against PrP shows that treatment of RK13-DC cells with increasing amounts of phosphoinositide phospholipase C (PI-PLC) increases the amount of detectable protein released into cell culture medium suggesting that mature PrP-NGLuc and PrP-CGLuc are anchored to the outer cell membrane by GPI. Additional lanes were excised for presentation purposes. ( c ) Densitometric quantification of the PrP signal from three different experiments as shown in ( b ). ( d ) Life cell immunofluorescence staining with the Sha31 antibody to PrP (left panels) shows that PrP-NGLuc and PrP-CGLuc are expressed in RK13-DC cells (top row), similar to wild-type PrP in N2a cells (bottom row). Bright-field microscopy (centre panels) shows the contours of the imaged cells. An overlay of fluorescence and bright-field images (right panels) shows that PrP-NGLuc, PrP-CGLuc, and PrP are located at the cell membrane. Bar = 20 µm. ( e ) The bioluminescence measured from RK13-DC cells expressing both PrP-NGLuc and PrP-CGLuc was more than 10-fold above background levels measured from RK13 cells stably expressing only PrP-NGLuc or PrP-CGLuc, or both NGLuc and CGLuc together each lacking the PrP moiety. A functional luciferase consisting of its N- and C-terminal halves was only formed in RK13-DC cells where dimers between the PrP moieties of PrP-NGLuc and PrP-CGLuc could form. In comparison to PrP-NGLuc and PrP-CGLuc in RK13-DC cells, bioluminescence of full-length GLuc in RK13 cells was almost 17-fold higher. In ( c , e ) error bars indicate SD.

    Techniques Used: Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Transfection, Molecular Weight, Planar Chromatography, Cell Culture, Immunofluorescence, Staining, Microscopy, Fluorescence, Functional Assay, Luciferase

    Compound screen identifies small molecules inhibiting PrP dimerization and conversion. ( a ) The Pharmacologically Active Compound Library (Selleckchem) was screened in RK13-DC cells for compounds reducing bioluminescence at a concentration of 10 µM after a 24-hour treatment. From among the 1,650 compounds 240 were found to reduce bioluminescence between 20–85%. From among these 73 compounds were found to reduce bioluminescence in a counter screen in RK13 cells stably expressing full-length Gaussia luciferase (RK13-GLuc cells), suggesting that they were either toxic or negatively affecting luciferase activity. From among the remaining 167 compounds, 13 among the most potent to reduce dimerization without negatively affecting bioluminescence of RK13-GLuc cells were selected for further analysis of their anti-prion activity in ScN2a cells. ( b ) The 13 selected compounds strongly reduced bioluminescence of RK13-DC cells by 60–80%. ( c ) From among these 13 compounds the most potent to reduce bioluminescence was JTC-801, a quinoline derivative. ( d ) The EC 50 for the reduction of bioluminescence of RK13-DC cells for JTC-801 was close to 2 µM, a concentration that did not affect cell survival within 24 h. ( e , f ) Biochemical and densitometric analysis shows that a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected ScN2a cells with an EC 50 of 370 nM. For presentation purposes additional lanes were excised from the image. Treatment of ScN2a cells with JTC-801 for 5 days did not affect cell for survival for concentrations ≤1.0 µM. ( g , h ) Similarly, a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected SMB cells with an EC 50 of 220 nM without being toxic for concentrations up to 1 µM. In ( b , d , f , h ) error bars indicate SD.
    Figure Legend Snippet: Compound screen identifies small molecules inhibiting PrP dimerization and conversion. ( a ) The Pharmacologically Active Compound Library (Selleckchem) was screened in RK13-DC cells for compounds reducing bioluminescence at a concentration of 10 µM after a 24-hour treatment. From among the 1,650 compounds 240 were found to reduce bioluminescence between 20–85%. From among these 73 compounds were found to reduce bioluminescence in a counter screen in RK13 cells stably expressing full-length Gaussia luciferase (RK13-GLuc cells), suggesting that they were either toxic or negatively affecting luciferase activity. From among the remaining 167 compounds, 13 among the most potent to reduce dimerization without negatively affecting bioluminescence of RK13-GLuc cells were selected for further analysis of their anti-prion activity in ScN2a cells. ( b ) The 13 selected compounds strongly reduced bioluminescence of RK13-DC cells by 60–80%. ( c ) From among these 13 compounds the most potent to reduce bioluminescence was JTC-801, a quinoline derivative. ( d ) The EC 50 for the reduction of bioluminescence of RK13-DC cells for JTC-801 was close to 2 µM, a concentration that did not affect cell survival within 24 h. ( e , f ) Biochemical and densitometric analysis shows that a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected ScN2a cells with an EC 50 of 370 nM. For presentation purposes additional lanes were excised from the image. Treatment of ScN2a cells with JTC-801 for 5 days did not affect cell for survival for concentrations ≤1.0 µM. ( g , h ) Similarly, a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected SMB cells with an EC 50 of 220 nM without being toxic for concentrations up to 1 µM. In ( b , d , f , h ) error bars indicate SD.

    Techniques Used: Concentration Assay, Stable Transfection, Expressing, Luciferase, Activity Assay, Infection

    12) Product Images from "A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization"

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32581-1

    PrP-NGLuc and PrP-CGLuc expression in RK13-DC cells results in bioluminescence. ( a ) Western blot analysis with the Sha31 antibody against PrP shows that RK13-DC cells stably express PrP-NGLuc and PrP-CGLuc from a bicistronic expression vector (lanes 3 and 4). Non-transfected RK13 cells do not express PrP and do not show a detectable signal for PrP (lanes 1 and 2), whereas N2a cells show the characteristic signal for wild-type PrP (lanes 5 and 6). Deglycosylation of cell lysates with peptide-N-glycosidase F (PNGase F, lanes 2, 4, and 6) resulted in lower molecular weight bands suggesting that mature PrP-NGLuc and PrP-CGLuc are properly glycosylated in RK13-DC cells as PrP is in N2a cells. Detection of tubulin on the same blot served as a loading control. Additional lanes were excised for presentation purposes. ( b ) Western blot analysis with the Sha31 antibody against PrP shows that treatment of RK13-DC cells with increasing amounts of phosphoinositide phospholipase C (PI-PLC) increases the amount of detectable protein released into cell culture medium suggesting that mature PrP-NGLuc and PrP-CGLuc are anchored to the outer cell membrane by GPI. Additional lanes were excised for presentation purposes. ( c ) Densitometric quantification of the PrP signal from three different experiments as shown in ( b ). ( d ) Life cell immunofluorescence staining with the Sha31 antibody to PrP (left panels) shows that PrP-NGLuc and PrP-CGLuc are expressed in RK13-DC cells (top row), similar to wild-type PrP in N2a cells (bottom row). Bright-field microscopy (centre panels) shows the contours of the imaged cells. An overlay of fluorescence and bright-field images (right panels) shows that PrP-NGLuc, PrP-CGLuc, and PrP are located at the cell membrane. Bar = 20 µm. ( e ) The bioluminescence measured from RK13-DC cells expressing both PrP-NGLuc and PrP-CGLuc was more than 10-fold above background levels measured from RK13 cells stably expressing only PrP-NGLuc or PrP-CGLuc, or both NGLuc and CGLuc together each lacking the PrP moiety. A functional luciferase consisting of its N- and C-terminal halves was only formed in RK13-DC cells where dimers between the PrP moieties of PrP-NGLuc and PrP-CGLuc could form. In comparison to PrP-NGLuc and PrP-CGLuc in RK13-DC cells, bioluminescence of full-length GLuc in RK13 cells was almost 17-fold higher. In ( c , e ) error bars indicate SD.
    Figure Legend Snippet: PrP-NGLuc and PrP-CGLuc expression in RK13-DC cells results in bioluminescence. ( a ) Western blot analysis with the Sha31 antibody against PrP shows that RK13-DC cells stably express PrP-NGLuc and PrP-CGLuc from a bicistronic expression vector (lanes 3 and 4). Non-transfected RK13 cells do not express PrP and do not show a detectable signal for PrP (lanes 1 and 2), whereas N2a cells show the characteristic signal for wild-type PrP (lanes 5 and 6). Deglycosylation of cell lysates with peptide-N-glycosidase F (PNGase F, lanes 2, 4, and 6) resulted in lower molecular weight bands suggesting that mature PrP-NGLuc and PrP-CGLuc are properly glycosylated in RK13-DC cells as PrP is in N2a cells. Detection of tubulin on the same blot served as a loading control. Additional lanes were excised for presentation purposes. ( b ) Western blot analysis with the Sha31 antibody against PrP shows that treatment of RK13-DC cells with increasing amounts of phosphoinositide phospholipase C (PI-PLC) increases the amount of detectable protein released into cell culture medium suggesting that mature PrP-NGLuc and PrP-CGLuc are anchored to the outer cell membrane by GPI. Additional lanes were excised for presentation purposes. ( c ) Densitometric quantification of the PrP signal from three different experiments as shown in ( b ). ( d ) Life cell immunofluorescence staining with the Sha31 antibody to PrP (left panels) shows that PrP-NGLuc and PrP-CGLuc are expressed in RK13-DC cells (top row), similar to wild-type PrP in N2a cells (bottom row). Bright-field microscopy (centre panels) shows the contours of the imaged cells. An overlay of fluorescence and bright-field images (right panels) shows that PrP-NGLuc, PrP-CGLuc, and PrP are located at the cell membrane. Bar = 20 µm. ( e ) The bioluminescence measured from RK13-DC cells expressing both PrP-NGLuc and PrP-CGLuc was more than 10-fold above background levels measured from RK13 cells stably expressing only PrP-NGLuc or PrP-CGLuc, or both NGLuc and CGLuc together each lacking the PrP moiety. A functional luciferase consisting of its N- and C-terminal halves was only formed in RK13-DC cells where dimers between the PrP moieties of PrP-NGLuc and PrP-CGLuc could form. In comparison to PrP-NGLuc and PrP-CGLuc in RK13-DC cells, bioluminescence of full-length GLuc in RK13 cells was almost 17-fold higher. In ( c , e ) error bars indicate SD.

    Techniques Used: Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Transfection, Molecular Weight, Planar Chromatography, Cell Culture, Immunofluorescence, Staining, Microscopy, Fluorescence, Functional Assay, Luciferase

    Compound screen identifies small molecules inhibiting PrP dimerization and conversion. ( a ) The Pharmacologically Active Compound Library (Selleckchem) was screened in RK13-DC cells for compounds reducing bioluminescence at a concentration of 10 µM after a 24-hour treatment. From among the 1,650 compounds 240 were found to reduce bioluminescence between 20–85%. From among these 73 compounds were found to reduce bioluminescence in a counter screen in RK13 cells stably expressing full-length Gaussia luciferase (RK13-GLuc cells), suggesting that they were either toxic or negatively affecting luciferase activity. From among the remaining 167 compounds, 13 among the most potent to reduce dimerization without negatively affecting bioluminescence of RK13-GLuc cells were selected for further analysis of their anti-prion activity in ScN2a cells. ( b ) The 13 selected compounds strongly reduced bioluminescence of RK13-DC cells by 60–80%. ( c ) From among these 13 compounds the most potent to reduce bioluminescence was JTC-801, a quinoline derivative. ( d ) The EC 50 for the reduction of bioluminescence of RK13-DC cells for JTC-801 was close to 2 µM, a concentration that did not affect cell survival within 24 h. ( e , f ) Biochemical and densitometric analysis shows that a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected ScN2a cells with an EC 50 of 370 nM. For presentation purposes additional lanes were excised from the image. Treatment of ScN2a cells with JTC-801 for 5 days did not affect cell for survival for concentrations ≤1.0 µM. ( g , h ) Similarly, a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected SMB cells with an EC 50 of 220 nM without being toxic for concentrations up to 1 µM. In ( b , d , f , h ) error bars indicate SD.
    Figure Legend Snippet: Compound screen identifies small molecules inhibiting PrP dimerization and conversion. ( a ) The Pharmacologically Active Compound Library (Selleckchem) was screened in RK13-DC cells for compounds reducing bioluminescence at a concentration of 10 µM after a 24-hour treatment. From among the 1,650 compounds 240 were found to reduce bioluminescence between 20–85%. From among these 73 compounds were found to reduce bioluminescence in a counter screen in RK13 cells stably expressing full-length Gaussia luciferase (RK13-GLuc cells), suggesting that they were either toxic or negatively affecting luciferase activity. From among the remaining 167 compounds, 13 among the most potent to reduce dimerization without negatively affecting bioluminescence of RK13-GLuc cells were selected for further analysis of their anti-prion activity in ScN2a cells. ( b ) The 13 selected compounds strongly reduced bioluminescence of RK13-DC cells by 60–80%. ( c ) From among these 13 compounds the most potent to reduce bioluminescence was JTC-801, a quinoline derivative. ( d ) The EC 50 for the reduction of bioluminescence of RK13-DC cells for JTC-801 was close to 2 µM, a concentration that did not affect cell survival within 24 h. ( e , f ) Biochemical and densitometric analysis shows that a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected ScN2a cells with an EC 50 of 370 nM. For presentation purposes additional lanes were excised from the image. Treatment of ScN2a cells with JTC-801 for 5 days did not affect cell for survival for concentrations ≤1.0 µM. ( g , h ) Similarly, a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected SMB cells with an EC 50 of 220 nM without being toxic for concentrations up to 1 µM. In ( b , d , f , h ) error bars indicate SD.

    Techniques Used: Concentration Assay, Stable Transfection, Expressing, Luciferase, Activity Assay, Infection

    13) Product Images from "A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture"

    Article Title: A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.15-18789

    MYOC secretion from NTM-5 cells parallels secretion from HEK-293T cells ( A ) GLuc luminescence assay of secreted MYOC eGLuc2 variants (same color designation as described in Fig. 6). Conditioned media aliquots (50 μL) from NTM-5 cells 48 hours post-transfection were assayed for the MYOC eGLuc2 fusion protein ( n = 3 independent experiments, ±SD, * P
    Figure Legend Snippet: MYOC secretion from NTM-5 cells parallels secretion from HEK-293T cells ( A ) GLuc luminescence assay of secreted MYOC eGLuc2 variants (same color designation as described in Fig. 6). Conditioned media aliquots (50 μL) from NTM-5 cells 48 hours post-transfection were assayed for the MYOC eGLuc2 fusion protein ( n = 3 independent experiments, ±SD, * P

    Techniques Used: Luminescence Assay, Transfection

    Enhanced MYOC secretion occurs independent of translation. ( A , B ) HEK-293T cells were transfected with WT, G244V, C245Y, G246R, or Y437H MYOC eGLuc2 for 48 hours at 37°C. The media was changed and cells were treated with 1 or 25 μM cycloheximide (CHX) at 37°C or 30°C for up to 24 hours. Conditioned media aliquots (50 μL) were assayed for MYOC eGLuc2 by the GLuc assay 6 ( A ) or 24 ( B ) hours after the temperature shift/CHX treatment ( n ≥ 3 independent experiments, ± SD, * P
    Figure Legend Snippet: Enhanced MYOC secretion occurs independent of translation. ( A , B ) HEK-293T cells were transfected with WT, G244V, C245Y, G246R, or Y437H MYOC eGLuc2 for 48 hours at 37°C. The media was changed and cells were treated with 1 or 25 μM cycloheximide (CHX) at 37°C or 30°C for up to 24 hours. Conditioned media aliquots (50 μL) were assayed for MYOC eGLuc2 by the GLuc assay 6 ( A ) or 24 ( B ) hours after the temperature shift/CHX treatment ( n ≥ 3 independent experiments, ± SD, * P

    Techniques Used: Transfection

    The GLuc assay can easily measure Y437H MYOC eGLuc2 levels before they are detectable by Western blotting. ( A ) HEK-293T cells were transfected for 48 hours followed by a media change with 2% FBS media. Thirty minutes to 1.5 hours after media change, conditioned media aliquots (20 μL) were taken and analyzed by the GLuc assay ( n = 3 independent experiments, ±SEM, * P
    Figure Legend Snippet: The GLuc assay can easily measure Y437H MYOC eGLuc2 levels before they are detectable by Western blotting. ( A ) HEK-293T cells were transfected for 48 hours followed by a media change with 2% FBS media. Thirty minutes to 1.5 hours after media change, conditioned media aliquots (20 μL) were taken and analyzed by the GLuc assay ( n = 3 independent experiments, ±SEM, * P

    Techniques Used: Western Blot, Transfection

    Analysis of pathogenic and nonpathogenic MYOC eGLuc2 mutants. ( A ) GLuc luminescence assay of secreted WT MYOC ( white bar ), predicted polymorphic (nonpathogenic) MYOC mutants ( gray bars , D208E, G244V), predicted pathogenic MYOC mutants ( black bars , C245Y, G246R, E300K, G434S, Y437H, Y471C) and an engineered mutant ( striped bar , Y437F), from transfected HEK-293T cells. At 48 hours after transfection, 50 μL of conditioned media was assayed for the MYOC eGLuc2 fusion protein. Media from untransfected cells was used as a control ( n ≥ 3 independent experiments, ±SD, ** P
    Figure Legend Snippet: Analysis of pathogenic and nonpathogenic MYOC eGLuc2 mutants. ( A ) GLuc luminescence assay of secreted WT MYOC ( white bar ), predicted polymorphic (nonpathogenic) MYOC mutants ( gray bars , D208E, G244V), predicted pathogenic MYOC mutants ( black bars , C245Y, G246R, E300K, G434S, Y437H, Y471C) and an engineered mutant ( striped bar , Y437F), from transfected HEK-293T cells. At 48 hours after transfection, 50 μL of conditioned media was assayed for the MYOC eGLuc2 fusion protein. Media from untransfected cells was used as a control ( n ≥ 3 independent experiments, ±SD, ** P

    Techniques Used: Luminescence Assay, Mutagenesis, Transfection

    14) Product Images from "A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization"

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32581-1

    Compound screen identifies small molecules inhibiting PrP dimerization and conversion. ( a ) The Pharmacologically Active Compound Library (Selleckchem) was screened in RK13-DC cells for compounds reducing bioluminescence at a concentration of 10 µM after a 24-hour treatment. From among the 1,650 compounds 240 were found to reduce bioluminescence between 20–85%. From among these 73 compounds were found to reduce bioluminescence in a counter screen in RK13 cells stably expressing full-length Gaussia luciferase (RK13-GLuc cells), suggesting that they were either toxic or negatively affecting luciferase activity. From among the remaining 167 compounds, 13 among the most potent to reduce dimerization without negatively affecting bioluminescence of RK13-GLuc cells were selected for further analysis of their anti-prion activity in ScN2a cells. ( b ) The 13 selected compounds strongly reduced bioluminescence of RK13-DC cells by 60–80%. ( c ) From among these 13 compounds the most potent to reduce bioluminescence was JTC-801, a quinoline derivative. ( d ) The EC 50 for the reduction of bioluminescence of RK13-DC cells for JTC-801 was close to 2 µM, a concentration that did not affect cell survival within 24 h. ( e , f ) Biochemical and densitometric analysis shows that a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected ScN2a cells with an EC 50 of 370 nM. For presentation purposes additional lanes were excised from the image. Treatment of ScN2a cells with JTC-801 for 5 days did not affect cell for survival for concentrations ≤1.0 µM. ( g , h ) Similarly, a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected SMB cells with an EC 50 of 220 nM without being toxic for concentrations up to 1 µM. In ( b , d , f , h ) error bars indicate SD.
    Figure Legend Snippet: Compound screen identifies small molecules inhibiting PrP dimerization and conversion. ( a ) The Pharmacologically Active Compound Library (Selleckchem) was screened in RK13-DC cells for compounds reducing bioluminescence at a concentration of 10 µM after a 24-hour treatment. From among the 1,650 compounds 240 were found to reduce bioluminescence between 20–85%. From among these 73 compounds were found to reduce bioluminescence in a counter screen in RK13 cells stably expressing full-length Gaussia luciferase (RK13-GLuc cells), suggesting that they were either toxic or negatively affecting luciferase activity. From among the remaining 167 compounds, 13 among the most potent to reduce dimerization without negatively affecting bioluminescence of RK13-GLuc cells were selected for further analysis of their anti-prion activity in ScN2a cells. ( b ) The 13 selected compounds strongly reduced bioluminescence of RK13-DC cells by 60–80%. ( c ) From among these 13 compounds the most potent to reduce bioluminescence was JTC-801, a quinoline derivative. ( d ) The EC 50 for the reduction of bioluminescence of RK13-DC cells for JTC-801 was close to 2 µM, a concentration that did not affect cell survival within 24 h. ( e , f ) Biochemical and densitometric analysis shows that a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected ScN2a cells with an EC 50 of 370 nM. For presentation purposes additional lanes were excised from the image. Treatment of ScN2a cells with JTC-801 for 5 days did not affect cell for survival for concentrations ≤1.0 µM. ( g , h ) Similarly, a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected SMB cells with an EC 50 of 220 nM without being toxic for concentrations up to 1 µM. In ( b , d , f , h ) error bars indicate SD.

    Techniques Used: Concentration Assay, Stable Transfection, Expressing, Luciferase, Activity Assay, Infection

    Related Articles

    Transfection:

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization
    Article Snippet: .. Transfected RK13 cells were analysed by fluorescence activated cell sorting (FACS) with the SAF32 antibody against PrP (Bertin Pharma) or the anti-GLuc antibody (New England Biolabs) against Gaussia luciferase for expression of PrP-NGLuc and PrP-CGLuc and similar constructs at the Flow Cytometry Core Facility of the University of Bonn using a BD FACSAria III (Becton Dickinson). .. RK13 cells expressing high surface protein levels were FACS sorted and maintained in 700 μg/mL G418 (Sigma) to obtain stably expressing RK13 cell lines.

    Luciferase:

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization
    Article Snippet: .. Transfected RK13 cells were analysed by fluorescence activated cell sorting (FACS) with the SAF32 antibody against PrP (Bertin Pharma) or the anti-GLuc antibody (New England Biolabs) against Gaussia luciferase for expression of PrP-NGLuc and PrP-CGLuc and similar constructs at the Flow Cytometry Core Facility of the University of Bonn using a BD FACSAria III (Becton Dickinson). .. RK13 cells expressing high surface protein levels were FACS sorted and maintained in 700 μg/mL G418 (Sigma) to obtain stably expressing RK13 cell lines.

    Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines
    Article Snippet: .. At 24 h post-transfection (p.t.), cells were analyzed for GFP expression and photographed using a fluorescent microscope (Olympus IX81) and camera (QIMAGING, Retiga 2000R); Gluc and Cluc expression levels were determined in tissue culture supernatants (TCS) using the BioluxGaussia and Cypridina Luciferase Assay kits (New England BioLabs) and quantified with a Lumicount luminometer (Packard). .. Reporter Gluc gene activation was normalized to that of Cluc and shown as fold induction over the level of induction of the negative control (no pDZ-NP).

    Article Title: RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii
    Article Snippet: .. To detect Gaussia luciferase, anti-gluc rabbit polyclonal antibody (E8023S; New England Biolabs) was used. ..

    Fluorescence:

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization
    Article Snippet: .. Transfected RK13 cells were analysed by fluorescence activated cell sorting (FACS) with the SAF32 antibody against PrP (Bertin Pharma) or the anti-GLuc antibody (New England Biolabs) against Gaussia luciferase for expression of PrP-NGLuc and PrP-CGLuc and similar constructs at the Flow Cytometry Core Facility of the University of Bonn using a BD FACSAria III (Becton Dickinson). .. RK13 cells expressing high surface protein levels were FACS sorted and maintained in 700 μg/mL G418 (Sigma) to obtain stably expressing RK13 cell lines.

    Construct:

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization
    Article Snippet: .. Transfected RK13 cells were analysed by fluorescence activated cell sorting (FACS) with the SAF32 antibody against PrP (Bertin Pharma) or the anti-GLuc antibody (New England Biolabs) against Gaussia luciferase for expression of PrP-NGLuc and PrP-CGLuc and similar constructs at the Flow Cytometry Core Facility of the University of Bonn using a BD FACSAria III (Becton Dickinson). .. RK13 cells expressing high surface protein levels were FACS sorted and maintained in 700 μg/mL G418 (Sigma) to obtain stably expressing RK13 cell lines.

    Flow Cytometry:

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization
    Article Snippet: .. Transfected RK13 cells were analysed by fluorescence activated cell sorting (FACS) with the SAF32 antibody against PrP (Bertin Pharma) or the anti-GLuc antibody (New England Biolabs) against Gaussia luciferase for expression of PrP-NGLuc and PrP-CGLuc and similar constructs at the Flow Cytometry Core Facility of the University of Bonn using a BD FACSAria III (Becton Dickinson). .. RK13 cells expressing high surface protein levels were FACS sorted and maintained in 700 μg/mL G418 (Sigma) to obtain stably expressing RK13 cell lines.

    Blocking Assay:

    Article Title: Arenavirus reverse genetics for vaccine development
    Article Snippet: .. After blocking for 1 h at room temperature with 10 % dry milk in 1× PBS, membranes were incubated with monoclonal primary antibodies against LCMV NP (1.1.3), Candid#1-NP (BEI Resources, KA03-AA01), a monoclonal antibody against GFP (Clontech, 632381), a polyclonal antibody against Gluc (New England Biolabs, E8023S), or a polyclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; AbCAM, AB9485) for 1 h at room temperature. .. Membranes were washed three times with 1× PBS containing 0.1 % Tween-20, and probed with secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin (Ig) antibodies (GE Healthcare) for 1 h at room temperature.

    Article Title: Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice
    Article Snippet: .. After blocking with 5% bovine serum albumin (BSA) in PBS, rabbit anti-gLuc antibody (New England Biolabs Inc., Madison, WI, USA) diluted 1:500 was dropped onto the grid, which was then incubated for 30 min. After washing with 0.5% BSA in PBS, the sample was incubated with a 10-nm protein A-gold conjugate (BBI Solutions, Cardiff, UK) for 20 min. .. The grid was washed with PBS and transferred to drop of 1% glutaraldehyde in PBS and incubated for 5 min.

    Cytometry:

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization
    Article Snippet: .. Transfected RK13 cells were analysed by fluorescence activated cell sorting (FACS) with the SAF32 antibody against PrP (Bertin Pharma) or the anti-GLuc antibody (New England Biolabs) against Gaussia luciferase for expression of PrP-NGLuc and PrP-CGLuc and similar constructs at the Flow Cytometry Core Facility of the University of Bonn using a BD FACSAria III (Becton Dickinson). .. RK13 cells expressing high surface protein levels were FACS sorted and maintained in 700 μg/mL G418 (Sigma) to obtain stably expressing RK13 cell lines.

    Acid Assay:

    Article Title: High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production
    Article Snippet: .. Lysates were cleared by centrifugation at 21.1 × 103 ×g 4 °C for 15 min, total protein was quantified using the bicinchoninic acid assay, and 2 mg of lysate was incubated with 5 μL of α-GLuc antibody (NEB) in 500 μL volume end-over-end at 4 °C. .. After 4 h, 30 μL of Protein A/G PLUS-Agarose bead slurry (Santa Cruz) was added and samples were incubated end-over-end at 4 °C overnight.

    Sandwich ELISA:

    Article Title: The Functionality of Minimal PiggyBac Transposons in Mammalian Cells
    Article Snippet: .. For sandwich enzyme-linked immunosorbent assay, rabbit polyclonal anti-GLuc antibody (Catalog No. E8023S, New England BioLabs (Ipswich, MA)) were bound to Costal Assay plate (96 well) black flat bottom polystyrene plate (Catalog No. 3925, Corning) and used to capture the recombinant GLuc. .. The collected serum and rabbit antimouse IgG HRP conjugated secondary antibody (Catalog No. 61–6520, Thermo Fisher Scientific, Waltham, MA) were used to probe captured recombinant GLuc by indirect enzyme-linked immunosorbent assay to compare anti-GLuc antibody level in mouse serum between samples.

    Incubation:

    Article Title: Arenavirus reverse genetics for vaccine development
    Article Snippet: .. After blocking for 1 h at room temperature with 10 % dry milk in 1× PBS, membranes were incubated with monoclonal primary antibodies against LCMV NP (1.1.3), Candid#1-NP (BEI Resources, KA03-AA01), a monoclonal antibody against GFP (Clontech, 632381), a polyclonal antibody against Gluc (New England Biolabs, E8023S), or a polyclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; AbCAM, AB9485) for 1 h at room temperature. .. Membranes were washed three times with 1× PBS containing 0.1 % Tween-20, and probed with secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin (Ig) antibodies (GE Healthcare) for 1 h at room temperature.

    Article Title: Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice
    Article Snippet: .. After blocking with 5% bovine serum albumin (BSA) in PBS, rabbit anti-gLuc antibody (New England Biolabs Inc., Madison, WI, USA) diluted 1:500 was dropped onto the grid, which was then incubated for 30 min. After washing with 0.5% BSA in PBS, the sample was incubated with a 10-nm protein A-gold conjugate (BBI Solutions, Cardiff, UK) for 20 min. .. The grid was washed with PBS and transferred to drop of 1% glutaraldehyde in PBS and incubated for 5 min.

    Article Title: High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production
    Article Snippet: .. Lysates were cleared by centrifugation at 21.1 × 103 ×g 4 °C for 15 min, total protein was quantified using the bicinchoninic acid assay, and 2 mg of lysate was incubated with 5 μL of α-GLuc antibody (NEB) in 500 μL volume end-over-end at 4 °C. .. After 4 h, 30 μL of Protein A/G PLUS-Agarose bead slurry (Santa Cruz) was added and samples were incubated end-over-end at 4 °C overnight.

    Microscopy:

    Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines
    Article Snippet: .. At 24 h post-transfection (p.t.), cells were analyzed for GFP expression and photographed using a fluorescent microscope (Olympus IX81) and camera (QIMAGING, Retiga 2000R); Gluc and Cluc expression levels were determined in tissue culture supernatants (TCS) using the BioluxGaussia and Cypridina Luciferase Assay kits (New England BioLabs) and quantified with a Lumicount luminometer (Packard). .. Reporter Gluc gene activation was normalized to that of Cluc and shown as fold induction over the level of induction of the negative control (no pDZ-NP).

    Expressing:

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization
    Article Snippet: .. Transfected RK13 cells were analysed by fluorescence activated cell sorting (FACS) with the SAF32 antibody against PrP (Bertin Pharma) or the anti-GLuc antibody (New England Biolabs) against Gaussia luciferase for expression of PrP-NGLuc and PrP-CGLuc and similar constructs at the Flow Cytometry Core Facility of the University of Bonn using a BD FACSAria III (Becton Dickinson). .. RK13 cells expressing high surface protein levels were FACS sorted and maintained in 700 μg/mL G418 (Sigma) to obtain stably expressing RK13 cell lines.

    Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines
    Article Snippet: .. At 24 h post-transfection (p.t.), cells were analyzed for GFP expression and photographed using a fluorescent microscope (Olympus IX81) and camera (QIMAGING, Retiga 2000R); Gluc and Cluc expression levels were determined in tissue culture supernatants (TCS) using the BioluxGaussia and Cypridina Luciferase Assay kits (New England BioLabs) and quantified with a Lumicount luminometer (Packard). .. Reporter Gluc gene activation was normalized to that of Cluc and shown as fold induction over the level of induction of the negative control (no pDZ-NP).

    FACS:

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization
    Article Snippet: .. Transfected RK13 cells were analysed by fluorescence activated cell sorting (FACS) with the SAF32 antibody against PrP (Bertin Pharma) or the anti-GLuc antibody (New England Biolabs) against Gaussia luciferase for expression of PrP-NGLuc and PrP-CGLuc and similar constructs at the Flow Cytometry Core Facility of the University of Bonn using a BD FACSAria III (Becton Dickinson). .. RK13 cells expressing high surface protein levels were FACS sorted and maintained in 700 μg/mL G418 (Sigma) to obtain stably expressing RK13 cell lines.

    Centrifugation:

    Article Title: High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production
    Article Snippet: .. Lysates were cleared by centrifugation at 21.1 × 103 ×g 4 °C for 15 min, total protein was quantified using the bicinchoninic acid assay, and 2 mg of lysate was incubated with 5 μL of α-GLuc antibody (NEB) in 500 μL volume end-over-end at 4 °C. .. After 4 h, 30 μL of Protein A/G PLUS-Agarose bead slurry (Santa Cruz) was added and samples were incubated end-over-end at 4 °C overnight.

    Recombinant:

    Article Title: The Functionality of Minimal PiggyBac Transposons in Mammalian Cells
    Article Snippet: .. For sandwich enzyme-linked immunosorbent assay, rabbit polyclonal anti-GLuc antibody (Catalog No. E8023S, New England BioLabs (Ipswich, MA)) were bound to Costal Assay plate (96 well) black flat bottom polystyrene plate (Catalog No. 3925, Corning) and used to capture the recombinant GLuc. .. The collected serum and rabbit antimouse IgG HRP conjugated secondary antibody (Catalog No. 61–6520, Thermo Fisher Scientific, Waltham, MA) were used to probe captured recombinant GLuc by indirect enzyme-linked immunosorbent assay to compare anti-GLuc antibody level in mouse serum between samples.

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    New England Biolabs gluc
    Contribution of PB2 and PB1 mutations of PR8/AA and PR8/Len in the viral polymerase activity at different temperatures. HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of the indicated combinations of ambisense pDZ expression plasmids encoding the PB2 and PB1 from PR8/WT, PR8/AA and PR8/Len together with the pDZ encoding PA and NP PR8/WT proteins; together with 500 ng of the hPol-I-GFP ( A ) and <t>-Gluc</t> ( B ), and 100 ng of SV40 <t>Cluc</t> to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and reporter gene expression was evaluated 24 h later by GFP imaging ( A ) and Gluc expression ( B ). Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that in the absence of pDZ NP plasmid. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P
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    Contribution of PB2 and PB1 mutations of PR8/AA and PR8/Len in the viral polymerase activity at different temperatures. HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of the indicated combinations of ambisense pDZ expression plasmids encoding the PB2 and PB1 from PR8/WT, PR8/AA and PR8/Len together with the pDZ encoding PA and NP PR8/WT proteins; together with 500 ng of the hPol-I-GFP ( A ) and -Gluc ( B ), and 100 ng of SV40 Cluc to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and reporter gene expression was evaluated 24 h later by GFP imaging ( A ) and Gluc expression ( B ). Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that in the absence of pDZ NP plasmid. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P

    Journal: Viruses

    Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

    doi: 10.3390/v11100928

    Figure Lengend Snippet: Contribution of PB2 and PB1 mutations of PR8/AA and PR8/Len in the viral polymerase activity at different temperatures. HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of the indicated combinations of ambisense pDZ expression plasmids encoding the PB2 and PB1 from PR8/WT, PR8/AA and PR8/Len together with the pDZ encoding PA and NP PR8/WT proteins; together with 500 ng of the hPol-I-GFP ( A ) and -Gluc ( B ), and 100 ng of SV40 Cluc to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and reporter gene expression was evaluated 24 h later by GFP imaging ( A ) and Gluc expression ( B ). Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that in the absence of pDZ NP plasmid. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P

    Article Snippet: At 24 h post-transfection (p.t.), cells were analyzed for GFP expression and photographed using a fluorescent microscope (Olympus IX81) and camera (QIMAGING, Retiga 2000R); Gluc and Cluc expression levels were determined in tissue culture supernatants (TCS) using the BioluxGaussia and Cypridina Luciferase Assay kits (New England BioLabs) and quantified with a Lumicount luminometer (Packard).

    Techniques: Activity Assay, Transfection, Expressing, Imaging, Plasmid Preparation

    Effect of temperature on the polymerase activity of PR8/AA and PR8/Len. ( A ) Schematic representation of segments 1 (PB2), 2 (PB1) and 8 (NS) of PR8/WT (white, left), PR8/AA (black, middle) and PR8/Len (grey, right). Amino acid substitutions to generate PR8/AA and PR8/Len are indicated. B-C) Minigenome activity: HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of ambisense pDZ expression plasmids encoding the minimal requirements for viral genome replication and gene transcription (PB2, PB1, PA, and NP) together with 500 ng of a vRNA-like expression plasmid encoding GFP ( B ) or Gluc ( C ) under the control of the human polymerase I promoter (hPol-I-GFP and -Gluc, respectively), and 100 ng of an SV40 Cluc expressing plasmid to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and viral replication and transcription was evaluated 24 h later by GFP ( B ) and luciferase ( C ) expression. Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that obtained in the absence of pDZ NP. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P

    Journal: Viruses

    Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

    doi: 10.3390/v11100928

    Figure Lengend Snippet: Effect of temperature on the polymerase activity of PR8/AA and PR8/Len. ( A ) Schematic representation of segments 1 (PB2), 2 (PB1) and 8 (NS) of PR8/WT (white, left), PR8/AA (black, middle) and PR8/Len (grey, right). Amino acid substitutions to generate PR8/AA and PR8/Len are indicated. B-C) Minigenome activity: HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of ambisense pDZ expression plasmids encoding the minimal requirements for viral genome replication and gene transcription (PB2, PB1, PA, and NP) together with 500 ng of a vRNA-like expression plasmid encoding GFP ( B ) or Gluc ( C ) under the control of the human polymerase I promoter (hPol-I-GFP and -Gluc, respectively), and 100 ng of an SV40 Cluc expressing plasmid to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and viral replication and transcription was evaluated 24 h later by GFP ( B ) and luciferase ( C ) expression. Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that obtained in the absence of pDZ NP. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P

    Article Snippet: At 24 h post-transfection (p.t.), cells were analyzed for GFP expression and photographed using a fluorescent microscope (Olympus IX81) and camera (QIMAGING, Retiga 2000R); Gluc and Cluc expression levels were determined in tissue culture supernatants (TCS) using the BioluxGaussia and Cypridina Luciferase Assay kits (New England BioLabs) and quantified with a Lumicount luminometer (Packard).

    Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Luciferase

    Schematic of minimal piggyBac mPB-GLuc-mCherry ( a ) and nontransposon GLuc-mCherry ( b ) bicistronic vectors. Both plasmids contained secretable Gaussia luciferase ( GLuc ) and mCherry genes separated with an internal ribosomal entry site (IRES) for synchronized expression from a cytomegalovirus (CMV) promoter and a bGH polyadenylation signal ( pA ) termination. Minimal terminal repeats ( 5′TRmin, 3′TRmin ) and the piggyBac transposase open reading frame (ORF) ( PBase ) with truncated terminal domains ( 5′TD ( trunc. ) , 3′TD ( trunc. )) was present only in mPB-GLuc-mCherry vector. PiggyBac transposase expression was controlled by the phosphoglycerate kinase (PGK) promoter as described for other minimal piggyBac plasmids. ( c ) Expression of mCherry in mice tails 3 days, 30 days, and 6 months after local injection/electroporation (arrows) of GLuc-mCherry and mPB-GLuc-mCherry vectors. Control mouse represents the background signal in a nontransfected mouse. ( d ) Indirect enzyme-linked immunosorbent assay (ELISA) showing production of antibodies against GLuc in mouse serum after a single delivery of either a nontransposon vector GLuc-mCherry or a minimal piggyBac vector mPB-GLuc-mCherry .

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

    doi: 10.1038/mtna.2016.76

    Figure Lengend Snippet: Schematic of minimal piggyBac mPB-GLuc-mCherry ( a ) and nontransposon GLuc-mCherry ( b ) bicistronic vectors. Both plasmids contained secretable Gaussia luciferase ( GLuc ) and mCherry genes separated with an internal ribosomal entry site (IRES) for synchronized expression from a cytomegalovirus (CMV) promoter and a bGH polyadenylation signal ( pA ) termination. Minimal terminal repeats ( 5′TRmin, 3′TRmin ) and the piggyBac transposase open reading frame (ORF) ( PBase ) with truncated terminal domains ( 5′TD ( trunc. ) , 3′TD ( trunc. )) was present only in mPB-GLuc-mCherry vector. PiggyBac transposase expression was controlled by the phosphoglycerate kinase (PGK) promoter as described for other minimal piggyBac plasmids. ( c ) Expression of mCherry in mice tails 3 days, 30 days, and 6 months after local injection/electroporation (arrows) of GLuc-mCherry and mPB-GLuc-mCherry vectors. Control mouse represents the background signal in a nontransfected mouse. ( d ) Indirect enzyme-linked immunosorbent assay (ELISA) showing production of antibodies against GLuc in mouse serum after a single delivery of either a nontransposon vector GLuc-mCherry or a minimal piggyBac vector mPB-GLuc-mCherry .

    Article Snippet: For sandwich enzyme-linked immunosorbent assay, rabbit polyclonal anti-GLuc antibody (Catalog No. E8023S, New England BioLabs (Ipswich, MA)) were bound to Costal Assay plate (96 well) black flat bottom polystyrene plate (Catalog No. 3925, Corning) and used to capture the recombinant GLuc.

    Techniques: Luciferase, Expressing, Plasmid Preparation, Mouse Assay, Injection, Electroporation, Indirect ELISA, Enzyme-linked Immunosorbent Assay

    Compound screen identifies small molecules inhibiting PrP dimerization and conversion. ( a ) The Pharmacologically Active Compound Library (Selleckchem) was screened in RK13-DC cells for compounds reducing bioluminescence at a concentration of 10 µM after a 24-hour treatment. From among the 1,650 compounds 240 were found to reduce bioluminescence between 20–85%. From among these 73 compounds were found to reduce bioluminescence in a counter screen in RK13 cells stably expressing full-length Gaussia luciferase (RK13-GLuc cells), suggesting that they were either toxic or negatively affecting luciferase activity. From among the remaining 167 compounds, 13 among the most potent to reduce dimerization without negatively affecting bioluminescence of RK13-GLuc cells were selected for further analysis of their anti-prion activity in ScN2a cells. ( b ) The 13 selected compounds strongly reduced bioluminescence of RK13-DC cells by 60–80%. ( c ) From among these 13 compounds the most potent to reduce bioluminescence was JTC-801, a quinoline derivative. ( d ) The EC 50 for the reduction of bioluminescence of RK13-DC cells for JTC-801 was close to 2 µM, a concentration that did not affect cell survival within 24 h. ( e , f ) Biochemical and densitometric analysis shows that a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected ScN2a cells with an EC 50 of 370 nM. For presentation purposes additional lanes were excised from the image. Treatment of ScN2a cells with JTC-801 for 5 days did not affect cell for survival for concentrations ≤1.0 µM. ( g , h ) Similarly, a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected SMB cells with an EC 50 of 220 nM without being toxic for concentrations up to 1 µM. In ( b , d , f , h ) error bars indicate SD.

    Journal: Scientific Reports

    Article Title: A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization

    doi: 10.1038/s41598-018-32581-1

    Figure Lengend Snippet: Compound screen identifies small molecules inhibiting PrP dimerization and conversion. ( a ) The Pharmacologically Active Compound Library (Selleckchem) was screened in RK13-DC cells for compounds reducing bioluminescence at a concentration of 10 µM after a 24-hour treatment. From among the 1,650 compounds 240 were found to reduce bioluminescence between 20–85%. From among these 73 compounds were found to reduce bioluminescence in a counter screen in RK13 cells stably expressing full-length Gaussia luciferase (RK13-GLuc cells), suggesting that they were either toxic or negatively affecting luciferase activity. From among the remaining 167 compounds, 13 among the most potent to reduce dimerization without negatively affecting bioluminescence of RK13-GLuc cells were selected for further analysis of their anti-prion activity in ScN2a cells. ( b ) The 13 selected compounds strongly reduced bioluminescence of RK13-DC cells by 60–80%. ( c ) From among these 13 compounds the most potent to reduce bioluminescence was JTC-801, a quinoline derivative. ( d ) The EC 50 for the reduction of bioluminescence of RK13-DC cells for JTC-801 was close to 2 µM, a concentration that did not affect cell survival within 24 h. ( e , f ) Biochemical and densitometric analysis shows that a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected ScN2a cells with an EC 50 of 370 nM. For presentation purposes additional lanes were excised from the image. Treatment of ScN2a cells with JTC-801 for 5 days did not affect cell for survival for concentrations ≤1.0 µM. ( g , h ) Similarly, a 5-day treatment with JTC-801 reduces the amount of PrP Sc in RML-infected SMB cells with an EC 50 of 220 nM without being toxic for concentrations up to 1 µM. In ( b , d , f , h ) error bars indicate SD.

    Article Snippet: Transfected RK13 cells were analysed by fluorescence activated cell sorting (FACS) with the SAF32 antibody against PrP (Bertin Pharma) or the anti-GLuc antibody (New England Biolabs) against Gaussia luciferase for expression of PrP-NGLuc and PrP-CGLuc and similar constructs at the Flow Cytometry Core Facility of the University of Bonn using a BD FACSAria III (Becton Dickinson).

    Techniques: Concentration Assay, Stable Transfection, Expressing, Luciferase, Activity Assay, Infection

    High-throughput screening strategy and results. (A) Schematic of screen design. Addition of 1 μg/mL doxycycline (Dox) to the stable single-colony Saos-2 GLuc.Col cell line induces expression of the eGLuc2.Colα2(I) fusion protein, which can be proteolytically processed post-secretion. Luminescence generated by the fusion protein may be assayed upon addition of the eGLuc2 substrate. (B) eGLuc2.Colα2(I) from Saos-2 GLuc.Col lysate was immunoprecipitated with an α-GLuc antibody and analyzed by immunoblotting, revealing that eGLuc2.Colα2(I) associates intracellularly with Colα1(I). (C) Disulfide-dependent assembly of eGLuc2.Colα2(I) with Colα1(I). Collagen-I precipitated from conditioned media of Saos-2 GLuc.Col cells was treated with iodoacetamide and heated in Laemmli buffer with or without DTT. Samples were separated by SDS-PAGE and analyzed by immunoblotting. (D) Plot of filtered screening results. (E) Validated small molecule modulators of collagen-I secretion.

    Journal: Biochemistry

    Article Title: High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production

    doi: 10.1021/acs.biochem.8b00378

    Figure Lengend Snippet: High-throughput screening strategy and results. (A) Schematic of screen design. Addition of 1 μg/mL doxycycline (Dox) to the stable single-colony Saos-2 GLuc.Col cell line induces expression of the eGLuc2.Colα2(I) fusion protein, which can be proteolytically processed post-secretion. Luminescence generated by the fusion protein may be assayed upon addition of the eGLuc2 substrate. (B) eGLuc2.Colα2(I) from Saos-2 GLuc.Col lysate was immunoprecipitated with an α-GLuc antibody and analyzed by immunoblotting, revealing that eGLuc2.Colα2(I) associates intracellularly with Colα1(I). (C) Disulfide-dependent assembly of eGLuc2.Colα2(I) with Colα1(I). Collagen-I precipitated from conditioned media of Saos-2 GLuc.Col cells was treated with iodoacetamide and heated in Laemmli buffer with or without DTT. Samples were separated by SDS-PAGE and analyzed by immunoblotting. (D) Plot of filtered screening results. (E) Validated small molecule modulators of collagen-I secretion.

    Article Snippet: Lysates were cleared by centrifugation at 21.1 × 103 ×g 4 °C for 15 min, total protein was quantified using the bicinchoninic acid assay, and 2 mg of lysate was incubated with 5 μL of α-GLuc antibody (NEB) in 500 μL volume end-over-end at 4 °C.

    Techniques: High Throughput Screening Assay, Expressing, Generated, Immunoprecipitation, SDS Page