prl-simian virus 40 (sv40) plasmid  (Promega)

Journal: Virology

Article Title: Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist

doi: 10.1016/j.virol.2009.11.033

Figure Lengend Snippet: PRRSV nsp1 proteins inhibit IFN-β production. (A, B) HEK293T cells cultured in 24-well plates were cotransfected with a plasmid expressing nsp1 proteins, a plasmid expressing influenza virus NS1, or pCAGGS empty vector (P), pRL-SV40, and a luciferase reporter plasmid p125-Luc (A) or pCIB-55-Luc (B). At 20 h post transfection, cells were infected with Sendai virus (SeV) for 16 h to stimulate the production of interferon. (C–H, J) HEK293T cells in 24-well plates were cotransfected with the plasmid pEFneo-RIG-I (C), pEFneo-MDA5 (D), pEGFP-IPS-1 (E), pEFneo-TBK1 (F), pEFneo-IKKɛ (G), or pCAGGS-IRF3 (H), or pcDNA3-TRIF (J), along with pRL-SV40, pCAGGS expressing nsp1 proteins, and pCIB-55 plasmid for 20–24 h. (I) HEK293T cells were cotransfected with pNF-kB-luc, pcDNA3-TRIF, pRL-SV40 and pCAGGS expressing nsp1 proteins or pCAGGS empty vector (P) for 20 h. Cells were harvested and measured for firefly and Renilla luciferase activities. Relative luciferase activity is defined as a ratio of firefly luciferase reporter activity to Renilla luciferase activity. Each data point shown represents a mean value from three experiments. Error bars show standard deviations of the normalized data.

Article Snippet: The pRL-SV40 plasmid that expresses a Renilla luciferase under the control of a simian virus (SV) 40 promoter was purchased from Promega (Madison, WI).

Techniques: Cell Culture, Plasmid Preparation, Expressing, Luciferase, Transfection, Infection, Activity Assay

Journal: Virology

Article Title: Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist

doi: 10.1016/j.virol.2009.11.033

Figure Lengend Snippet: PRRSV nsp1 proteins inhibit expression from an ISRE promoter. HEK293T cells were cotransfected with pISRE-luc, pRL-SV40 and pCAGGS expressing nsp1 proteins or pCAGGS empty vector (P) for 20 h. Cells were then infected with Sendai virus (A) or treated with IFN-α (B) and IFN-β (C) for 20 h. The cells were harvested and measured for firefly and Renilla luciferase activities. Relative luciferase activity is defined as a ratio of firefly luciferase reporter activity to Renilla luciferase activity. Each data point shown represents a mean value from three experiments. Error bars show standard deviations of the normalized data.

Article Snippet: The pRL-SV40 plasmid that expresses a Renilla luciferase under the control of a simian virus (SV) 40 promoter was purchased from Promega (Madison, WI).

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

Journal:

Article Title: Requirement of p53 targets in chemosensitization of colonic carcinoma to death ligand therapy

doi: 10.1073/pnas.2435285100

Figure Lengend Snippet: p53 is required for sensitizing Bax-/- cells to TRAIL. Bax-/- HCT116 cells were infected with Ad-E6 or Ad-LacZ for 16 h. The infected cells were treated as described in the Fig. 1 legend. (A) p53 protein was determined by Western blot and active caspase 3 was analyzed by flow cytometry. (B) Infected cells were transfected with PG13-Luc and pRL-SV40 plasmids. Luciferase activity was determined by Fisher luminometer (Lower). PG13-Luc and pRL-SV40 stably transfected Bax-/- HCT116 cells were treated as described above, images were obtained by using the cooled charge-coupled camera of the in vivo imaging system (Upper).

Article Snippet: The PG13-luc, a p53 reporter with a firefly luciferase gene under the control of 13 p53 response elements, has been described ( 11 ). pRL-SV40 (simian virus 40) vector was purchased from Promega, and this vector contains a cDNA encoding Renilla luciferase, which was originally cloned from the marine organism Renilla reniformis .

Techniques: Infection, Western Blot, Flow Cytometry, Transfection, Luciferase, Activity Assay, Stable Transfection, In Vivo Imaging

Journal:

Article Title: Requirement of p53 targets in chemosensitization of colonic carcinoma to death ligand therapy

doi: 10.1073/pnas.2435285100

Figure Lengend Snippet: Optical imaging of p53 activity in living mice. PG13-Luc and pRL-SV40 stably transfected Bax-/- HCT116 cells were infected with Ad-LacZ or Ad-E6 for 16 h. Cells (1 × 106) were s.c. implanted into left or right forearm. CPT11 (80 mg/kg) was administered by i.p. injection. After 0 or 16 h of treatment, coelenterazine was injected 3 min before imaging, 2 h apart, followed by d-luciferin injection (5 min before imaging). A whole body image was acquired by using the cooled charge-coupled device camera. Each image was acquired at the same time, relative to the injected substrate, and all of the images are shown at the same scale.

Article Snippet: The PG13-luc, a p53 reporter with a firefly luciferase gene under the control of 13 p53 response elements, has been described ( 11 ). pRL-SV40 (simian virus 40) vector was purchased from Promega, and this vector contains a cDNA encoding Renilla luciferase, which was originally cloned from the marine organism Renilla reniformis .

Techniques: Optical Imaging, Activity Assay, Stable Transfection, Transfection, Infection, Injection, Imaging

Journal:

Article Title: Interferon Regulatory Factor 3 Is Required for Viral Induction of Beta Interferon in Primary Cardiac Myocyte Cultures

doi:

Figure Lengend Snippet: Reovirus induces an IFN-β reporter construct, pβLux. L929 cells or primary cardiac myocyte cultures were transfected with the indicated plasmid and the normalization plasmid pRL-SV40 and infected 1 day posttransfection. The cells or cultures were mock infected (▨) or infected with reovirus T3D (■). Luciferase activity was measured 18 to 20 h postinfection. For each well, normalized luciferase activity was determined by dividing firefly luciferase activity by renilla luciferase activity. Each bar shows the mean of three wells (or four wells for L929 cells) (each error bar shows the standard error of the mean). Similar results were obtained in replicate experiments. Asterisks denote a significant increase between mock- and virus-infected cultures (for L929 cells, P < 0.001; for cardiac myocytes, P = 0.011).

Article Snippet: The PCR fragment was gel purified and inserted into pGL3-Basic (containing the firefly luciferase gene but no promoter; Promega, Madison, Wis.) by using Hin dIII restriction sites. pGL3-Basic (lacking a promoter and therefore expressing baseline firefly luciferase), pGL3-Control (expressing firefly luciferase constitutively from a simian virus 40 promoter), and pRL-SV40 (expressing renilla luciferase constitutively from a simian virus 40 promoter) were all purchased (Promega). pEF-HAIRF-3 (expressing IRF-3) and pEF-HAIRF-3 58–427 (expressing a dominant negative IRF-3) contain the human EF-1α promoter for constitutive expression ( 30 , 51 ) and were generously provided by Takashi Fujita (Kyoto University, Kyoto, Japan). pEF-BOS, a control plasmid, was constructed by removing IRF-3 from pEF-HAIRF-3 by Eco RI restriction digestion followed by ligation of the remaining fragment. pBOSLux was constructed by using PCR to add Xba I sites to the luciferase gene of pGL3-Control and then inserting the PCR product into pEF-BOS, using Xba I restriction sites.

Techniques: Construct, Transfection, Plasmid Preparation, Infection, Luciferase, Activity Assay

Journal:

Article Title: Interferon Regulatory Factor 3 Is Required for Viral Induction of Beta Interferon in Primary Cardiac Myocyte Cultures

doi:

Figure Lengend Snippet: pβLux activity is induced in cells overexpressing IRF-3. L929 cells or primary cardiac myocyte cultures were transfected with pβLux, the normalization plasmid pRL-SV40, and the indicated amounts of either control DNA (pEF-BOS) (▨) or a plasmid expressing IRF-3 (■). Cells were infected 1 day posttransfection, and luciferase activity was measured 18 to 20 h postinfection. For each well, normalized luciferase activity was determined by dividing firefly luciferase activity by renilla luciferase activity. Each bar shows the mean of three wells (each error bar shows the standard error of the mean). Similar results were obtained in replicate experiments. Asterisks denote significant increases between cultures transfected with control DNA or IRF-3 (for L929 cells, from left to right, P = 0.046, 0.009, 0.002, <0.001, and < 0.001; for cardiac myocytes, from left to right, P = 0.001, 0.049, 0.002, and <0.001).

Article Snippet: The PCR fragment was gel purified and inserted into pGL3-Basic (containing the firefly luciferase gene but no promoter; Promega, Madison, Wis.) by using Hin dIII restriction sites. pGL3-Basic (lacking a promoter and therefore expressing baseline firefly luciferase), pGL3-Control (expressing firefly luciferase constitutively from a simian virus 40 promoter), and pRL-SV40 (expressing renilla luciferase constitutively from a simian virus 40 promoter) were all purchased (Promega). pEF-HAIRF-3 (expressing IRF-3) and pEF-HAIRF-3 58–427 (expressing a dominant negative IRF-3) contain the human EF-1α promoter for constitutive expression ( 30 , 51 ) and were generously provided by Takashi Fujita (Kyoto University, Kyoto, Japan). pEF-BOS, a control plasmid, was constructed by removing IRF-3 from pEF-HAIRF-3 by Eco RI restriction digestion followed by ligation of the remaining fragment. pBOSLux was constructed by using PCR to add Xba I sites to the luciferase gene of pGL3-Control and then inserting the PCR product into pEF-BOS, using Xba I restriction sites.

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

Journal:

Article Title: Interferon Regulatory Factor 3 Is Required for Viral Induction of Beta Interferon in Primary Cardiac Myocyte Cultures

doi:

Figure Lengend Snippet: IRF-3 is not required for but regulates pβLux induction in L929 cells. (A) L929 cells were transfected with pβLux, the normalization plasmid pRL-SV40, the indicated plasmid (IRF-3 and/or Dom.Neg. IRF-3), and/or control DNA (pEF-BOS) for a constant plasmid DNA concentration of 7.32 μg per well. Cells were infected 1 day posttransfection, and luciferase activity was measured 18 to 20 h postinfection. The cells were mock infected (▨) or infected with reovirus T3D (■). For each well, normalized luciferase activity was determined by dividing firefly luciferase activity by renilla luciferase activity. Each bar shows the mean of three wells (each error bar shows the standard error of the mean). (B) L929 cells were transfected with pβLux, the normalization plasmid pRL-SV40, and the indicated amounts of control DNA (pEF-BOS) or a plasmid expressing a dominant negative IRF-3 protein. Cells were infected, and luciferase activity was measured as described above. Fold inhibition was calculated by dividing normalized luciferase activity from virally infected cells transfected with control DNA by those transfected with dominant negative IRF-3. Each datum point is expressed as the average fold inhibition of three wells ± standard error of the mean. The results of two separate experiments are shown by the two lines, with average values of viral induction of pβLux of 9- and 11-fold. (C) L929 cells were transfected with the normalization plasmid pRL-SV40 and the indicated amounts of pBOSLux, mock infected, and harvested for luciferase as described above. For two separate experiments (depicted by the two lines), each datum point is expressed as 103 times the mean of three wells ± standard error of the mean.

Article Snippet: The PCR fragment was gel purified and inserted into pGL3-Basic (containing the firefly luciferase gene but no promoter; Promega, Madison, Wis.) by using Hin dIII restriction sites. pGL3-Basic (lacking a promoter and therefore expressing baseline firefly luciferase), pGL3-Control (expressing firefly luciferase constitutively from a simian virus 40 promoter), and pRL-SV40 (expressing renilla luciferase constitutively from a simian virus 40 promoter) were all purchased (Promega). pEF-HAIRF-3 (expressing IRF-3) and pEF-HAIRF-3 58–427 (expressing a dominant negative IRF-3) contain the human EF-1α promoter for constitutive expression ( 30 , 51 ) and were generously provided by Takashi Fujita (Kyoto University, Kyoto, Japan). pEF-BOS, a control plasmid, was constructed by removing IRF-3 from pEF-HAIRF-3 by Eco RI restriction digestion followed by ligation of the remaining fragment. pBOSLux was constructed by using PCR to add Xba I sites to the luciferase gene of pGL3-Control and then inserting the PCR product into pEF-BOS, using Xba I restriction sites.

Techniques: Transfection, Plasmid Preparation, Concentration Assay, Infection, Luciferase, Activity Assay, Expressing, Dominant Negative Mutation, Inhibition

Journal:

Article Title: Interferon Regulatory Factor 3 Is Required for Viral Induction of Beta Interferon in Primary Cardiac Myocyte Cultures

doi:

Figure Lengend Snippet: IRF-3 is required for reovirus induction of pβLux in primary cardiac myocyte cultures. Primary cardiac myocyte cultures were transfected with pβLux, the normalization plasmid pRL-SV40, and control DNA (pEF-BOS) or a plasmid expressing a dominant negative (Dom.Neg.) IRF-3 protein. Cells were infected 1 day posttransfection, and luciferase activity was measured 18 to 20 h postinfection. The cells were mock infected (▨) or infected with reovirus T3D (■). Results from three independent experiments are shown. For each well, normalized luciferase activity was determined by dividing firefly luciferase activity by renilla luciferase activity. Each bar shows the mean of three wells (each error bar shows the standard error of the mean). Asterisks denote no significant difference between virus- and mock-infected cultures (P > 0.05 for all three experiments).

Article Snippet: The PCR fragment was gel purified and inserted into pGL3-Basic (containing the firefly luciferase gene but no promoter; Promega, Madison, Wis.) by using Hin dIII restriction sites. pGL3-Basic (lacking a promoter and therefore expressing baseline firefly luciferase), pGL3-Control (expressing firefly luciferase constitutively from a simian virus 40 promoter), and pRL-SV40 (expressing renilla luciferase constitutively from a simian virus 40 promoter) were all purchased (Promega). pEF-HAIRF-3 (expressing IRF-3) and pEF-HAIRF-3 58–427 (expressing a dominant negative IRF-3) contain the human EF-1α promoter for constitutive expression ( 30 , 51 ) and were generously provided by Takashi Fujita (Kyoto University, Kyoto, Japan). pEF-BOS, a control plasmid, was constructed by removing IRF-3 from pEF-HAIRF-3 by Eco RI restriction digestion followed by ligation of the remaining fragment. pBOSLux was constructed by using PCR to add Xba I sites to the luciferase gene of pGL3-Control and then inserting the PCR product into pEF-BOS, using Xba I restriction sites.

Techniques: Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Infection, Luciferase, Activity Assay

Journal:

Article Title: Role of Secondary Structure in Discrimination between Constitutive and Inducible Activators

doi:

Figure Lengend Snippet: Characterization of a minimal transactivation domain in c-Myb that binds to the KIX domain of CBP. (A) Mammalian two-hybrid assays performed by using Gal4-KIX and c-Myb/VP16 expression vectors to examine interactions of c-Myb with CBP. COS-7 cells were transfected with 5×Gal4-Luc and pRL-SV40 Renilla luciferase reporters and with expression vectors for Gal-KIX and 2×VP16-Myb fragments. The hatched box represents the minimal KIX interaction domain of c-Myb encompassing aa 290 to 315. Inclusive amino acid endpoints of c-Myb fragments fused to tandem VP16 activation domains are indicated. Results are shown as fold activation (mean ± standard deviation, n = 2) over that by empty RSV expression vector (without VP16-Myb). c-Myb/VP16 polypeptides were expressed at comparable levels in transfected cells (data not shown). Inclusive endpoints of c-Myb are shown. (B) c-Myb (aa 290 to 315) stimulates target gene expression when fused to the Gal4 DNA binding domain (DBD). 293 cells were transfected with 5×Gal Luc and RSV-β-Gal reporter plasmids as well as vectors expressing Gal4 DBD or Gal-Myb (aa 290 to 315). Activity is reported as fold activation over that by Gal DBD (mean ± standard deviation, n = 3).

Article Snippet: For the mammalian two-hybrid assays, COS-7 cells in 3.5-cm-diameter dishes were transfected with 0.5 μg of 5×Gal-Luc reporter plasmid, 0.5 μg of Gal-KIX S/B expression plasmid, and 1.5 μg of RSV2×VP16-Myb, RSV-Myb, or pGR (RSV) expression plasmid and 0.01 μg of pRL-SV40 (SV40, simian virus 40) (Promega) by the calcium phosphate method.

Techniques: Expressing, Transfection, Luciferase, Activation Assay, Standard Deviation, Plasmid Preparation, Binding Assay, Activity Assay