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Promega dual specific luciferase assay kit
DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before <t>luciferase</t> assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a <t>dual-specific</t> luciferase <t>assay</t> <t>kit.</t> (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  
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1) Product Images from "Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway"

Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway

Journal: Journal of Virology

doi: 10.1128/JVI.00588-19

DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  
Figure Legend Snippet: DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  

Techniques Used: Inhibition, Transfection, Infection, Luciferase, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.
Figure Legend Snippet: FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.

Techniques Used: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Infection, Western Blot, Translocation Assay, Clear Native PAGE, Staining, Confocal Microscopy

2) Product Images from "Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway"

Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway

Journal: Journal of Virology

doi: 10.1128/JVI.00588-19

DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋅C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P
Figure Legend Snippet: DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋅C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P

Techniques Used: Inhibition, Transfection, Infection, Luciferase, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.
Figure Legend Snippet: FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.

Techniques Used: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Infection, Western Blot, Translocation Assay, Clear Native PAGE, Staining, Confocal Microscopy

3) Product Images from "Foot-and-Mouth Disease Virus Viroporin 2B Antagonizes RIG-I-Mediated Antiviral Effects by Inhibition of Its Protein Expression"

Article Title: Foot-and-Mouth Disease Virus Viroporin 2B Antagonizes RIG-I-Mediated Antiviral Effects by Inhibition of Its Protein Expression

Journal: Journal of Virology

doi: 10.1128/JVI.01310-16

2B protein induces the reduction of RIG-I and suppresses RIG-I-mediated signal transduction. (A) PK-15 cells (5 × 10 5 cells in each well) were grown in six-well plates, and the monolayer cells were transfected with different doses of Flag-2B-expressing plasmids (0, 0.5, 1, or 2 μg). The expression of endogenous RIG-I and Flag-2B proteins was detected by Western blotting at 48 hpt. RIG-I was detected by using rabbit anti-RIG-I polyclonal antibody. (B) PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg). The cells were collected at 0, 4, 8, 12, 24, 36, and 48 hpt, and the cell lysates were analyzed by Western blotting to detect the expression levels of RIG-I and any possible cleaved bands. (C) PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg) or empty vector (2 μg) in the presence or absence of 5′ppp-dsRNA control or 5′ppp-dsRNA (1 μg/ml; InvivoGen). The expression of two ISGs (ISG15 and GBP1) was determined by qPCR assay at 24 hpt. The data represent results from one of the triplicate experiments. (D) HEK293T cells (5 × 10 5 cells in each well) were transfected with HA-RIG-I-, HA-VISA-, HA-TBK1-, HA-MITA-, or HA-IRF3-expressing plasmids (2 μg), along with Flag-2B-expressing plasmid or empty vector (2 μg). The expression of HA-RIG-I, HA-VISA, HA-TBK1, HA-MITA, HA-IRF3, and Flag-2B was detected by Western blotting at 48 hpt. Mouse anti-HA antibody was used to detect HA-tagged proteins. (E) HEK293T cells (10 5 cells in each well) were seeded in 24-well plates, and the monolayer cells were transfected with Flag-2B-expressing plasmid (0.1 μg) or empty vector (0.1 μg), along with IFN-β luciferase reporter plasmid (0.05 μg). pRL-TK Renilla luciferase reporter plasmid (0.01 μg) was used in the reporter assay to normalize the transfection efficiency. At 24 h after transfection, the cells were left infected or uninfected with SeV (100 HAU/ml) for 16 h. A dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and Renilla . Empty vector plasmid was used in the transfection process to ensure that the cells received the same amounts of total plasmids. The data represent the means and standard deviations from three independent experiments. (F) HEK293T cells (10 5 cells in each well) were cotransfected with HA-RIG-I-expressing plasmid (0.1 μg) or empty vector (0.1 μg) and Flag-2B-expressing plasmid (0.1 μg) or empty Flag vector (0.1 μg), along with IFN-β luciferase reporter plasmid (0.1 μg). The pRL-TK Renilla luciferase reporter plasmid (0.01 μg) was used in the reporter assay to normalize the transfection efficiency. The dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and Renilla at 24 hpt as described for panel E. (G) FMDV 2B enhances virus replication in infected cells. PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg) or empty vector (2 μg). At 24 h after transfection, the cells were infected or uninfected with FMDV (MOI of 0.5) for 12 h. The expression of viral RNA was determined by qPCR assay. The viral VP1 proteins were detected by Western blotting. (H) FMDV 2B, L pro , and 3C pro enhance virus replication in infected cells. PK-15 cells (5 × 10 5 cells in each well) were transfected with empty vector, Flag-2B-, Flag-3C-, or Flag-L-expressing plasmids (2 μg). At 24 h after transfection, the cells were infected with FMDV (MOI of 0.5) for 12 h. The viral titers were determined by using a TCID 50 assay. All of the above-described experiments were repeated three times, with similar results. ** , P
Figure Legend Snippet: 2B protein induces the reduction of RIG-I and suppresses RIG-I-mediated signal transduction. (A) PK-15 cells (5 × 10 5 cells in each well) were grown in six-well plates, and the monolayer cells were transfected with different doses of Flag-2B-expressing plasmids (0, 0.5, 1, or 2 μg). The expression of endogenous RIG-I and Flag-2B proteins was detected by Western blotting at 48 hpt. RIG-I was detected by using rabbit anti-RIG-I polyclonal antibody. (B) PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg). The cells were collected at 0, 4, 8, 12, 24, 36, and 48 hpt, and the cell lysates were analyzed by Western blotting to detect the expression levels of RIG-I and any possible cleaved bands. (C) PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg) or empty vector (2 μg) in the presence or absence of 5′ppp-dsRNA control or 5′ppp-dsRNA (1 μg/ml; InvivoGen). The expression of two ISGs (ISG15 and GBP1) was determined by qPCR assay at 24 hpt. The data represent results from one of the triplicate experiments. (D) HEK293T cells (5 × 10 5 cells in each well) were transfected with HA-RIG-I-, HA-VISA-, HA-TBK1-, HA-MITA-, or HA-IRF3-expressing plasmids (2 μg), along with Flag-2B-expressing plasmid or empty vector (2 μg). The expression of HA-RIG-I, HA-VISA, HA-TBK1, HA-MITA, HA-IRF3, and Flag-2B was detected by Western blotting at 48 hpt. Mouse anti-HA antibody was used to detect HA-tagged proteins. (E) HEK293T cells (10 5 cells in each well) were seeded in 24-well plates, and the monolayer cells were transfected with Flag-2B-expressing plasmid (0.1 μg) or empty vector (0.1 μg), along with IFN-β luciferase reporter plasmid (0.05 μg). pRL-TK Renilla luciferase reporter plasmid (0.01 μg) was used in the reporter assay to normalize the transfection efficiency. At 24 h after transfection, the cells were left infected or uninfected with SeV (100 HAU/ml) for 16 h. A dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and Renilla . Empty vector plasmid was used in the transfection process to ensure that the cells received the same amounts of total plasmids. The data represent the means and standard deviations from three independent experiments. (F) HEK293T cells (10 5 cells in each well) were cotransfected with HA-RIG-I-expressing plasmid (0.1 μg) or empty vector (0.1 μg) and Flag-2B-expressing plasmid (0.1 μg) or empty Flag vector (0.1 μg), along with IFN-β luciferase reporter plasmid (0.1 μg). The pRL-TK Renilla luciferase reporter plasmid (0.01 μg) was used in the reporter assay to normalize the transfection efficiency. The dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and Renilla at 24 hpt as described for panel E. (G) FMDV 2B enhances virus replication in infected cells. PK-15 cells (5 × 10 5 cells in each well) were transfected with Flag-2B-expressing plasmid (2 μg) or empty vector (2 μg). At 24 h after transfection, the cells were infected or uninfected with FMDV (MOI of 0.5) for 12 h. The expression of viral RNA was determined by qPCR assay. The viral VP1 proteins were detected by Western blotting. (H) FMDV 2B, L pro , and 3C pro enhance virus replication in infected cells. PK-15 cells (5 × 10 5 cells in each well) were transfected with empty vector, Flag-2B-, Flag-3C-, or Flag-L-expressing plasmids (2 μg). At 24 h after transfection, the cells were infected with FMDV (MOI of 0.5) for 12 h. The viral titers were determined by using a TCID 50 assay. All of the above-described experiments were repeated three times, with similar results. ** , P

Techniques Used: Transduction, Transfection, Expressing, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Infection

4) Product Images from "Cellular RNA Helicase DDX1 Is Involved in Transmissible Gastroenteritis Virus nsp14-Induced Interferon-Beta Production"

Article Title: Cellular RNA Helicase DDX1 Is Involved in Transmissible Gastroenteritis Virus nsp14-Induced Interferon-Beta Production

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00940

Involvement of DDX1 in the antiviral response triggered by transmissible gastroenteritis virus (TGEV). (A,B) Assessment of the silencing efficiency of psiDDX1. PK-15 cells were transfected with 50 nM/well of psiDDX1 or siNC for 36 h each. The expression levels of porcine DDX1 were determined by RT-qPCR (A) and western blot assay (B) . (C,D) PK-15 cells were cotransfected with psiDDX1 or siNC, along with pRL-TK, IFN-β-Luc (C) , or NF-κB-Luc (D) . At 12 h post-transfection, cells were mock infected or infected with TGEV [multiplicity of infection (MOI) = 0.5]. Dual luciferase assays were performed at 24 hpi. (E) PK-15 cells were transfected with psiDDX1 or siNC, and 12 h later, cells were mock infected or infected with TGEV (MOI = 0.5) for 24 h. Then, the cells were collected for western blot assay with specific antibodies against p65, p-p65, DDX1, or TGEV N protein, using β-actin expression as a loading control. The ratio of phosphorylated/total p65 was analyzed using ImageJ Software. (F–J) PK-15 cells were treated as described for (E) and collected at 24 hpi. Cell RNAs were extracted for RT-qPCR to examine the mRNA expression levels of IFIT1 (F) , IFIT2 (G) , IFIT3 (H) , IL-6 (I) , and IL-8 (J) . The mRNA expression levels were normalized to porcine GAPDH transcripts. Values are the mean ± SD of three independent tests. * P
Figure Legend Snippet: Involvement of DDX1 in the antiviral response triggered by transmissible gastroenteritis virus (TGEV). (A,B) Assessment of the silencing efficiency of psiDDX1. PK-15 cells were transfected with 50 nM/well of psiDDX1 or siNC for 36 h each. The expression levels of porcine DDX1 were determined by RT-qPCR (A) and western blot assay (B) . (C,D) PK-15 cells were cotransfected with psiDDX1 or siNC, along with pRL-TK, IFN-β-Luc (C) , or NF-κB-Luc (D) . At 12 h post-transfection, cells were mock infected or infected with TGEV [multiplicity of infection (MOI) = 0.5]. Dual luciferase assays were performed at 24 hpi. (E) PK-15 cells were transfected with psiDDX1 or siNC, and 12 h later, cells were mock infected or infected with TGEV (MOI = 0.5) for 24 h. Then, the cells were collected for western blot assay with specific antibodies against p65, p-p65, DDX1, or TGEV N protein, using β-actin expression as a loading control. The ratio of phosphorylated/total p65 was analyzed using ImageJ Software. (F–J) PK-15 cells were treated as described for (E) and collected at 24 hpi. Cell RNAs were extracted for RT-qPCR to examine the mRNA expression levels of IFIT1 (F) , IFIT2 (G) , IFIT3 (H) , IL-6 (I) , and IL-8 (J) . The mRNA expression levels were normalized to porcine GAPDH transcripts. Values are the mean ± SD of three independent tests. * P

Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Infection, Luciferase, Software

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Article Snippet: Paragraph title: Dual-luciferase reporter assay ... At 24 h after transfection or further processing, dual-luciferase assays were performed using a dual-specific luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

Infection:

Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway
Article Snippet: .. At 24 hpt, the cells were mock infected or infected with SeV for 12 h; the luciferase activity was then measured by using a dual-specific luciferase assay kit (Promega). ..

Incubation:

Article Title: Nobiletin alleviates endometriosis via down-regulating NF-κB activity in endometriosis mouse model
Article Snippet: After 34 h of transfection, cells were incubated with nobiletin of 10 and 20 µg/ml for 2 h respectively. .. Luciferase assays were performed with a dual-specific luciferase assay kit (Promega, Durham, NC).

Activity Assay:

Article Title: USP13 negatively regulates antiviral responses by deubiquitinating STING
Article Snippet: After 24 h, luciferase assays were performed with a dual-specific luciferase reporter kit (Promega). .. The activity of firefly luciferase was normalized by that of Renilla luciferase to obtain relative luciferase activity.

Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway
Article Snippet: .. At 24 hpt, the cells were mock infected or infected with SeV for 12 h; the luciferase activity was then measured by using a dual-specific luciferase assay kit (Promega). ..

Expressing:

Article Title: Nobiletin alleviates endometriosis via down-regulating NF-κB activity in endometriosis mouse model
Article Snippet: Luciferase assay The HEK293 cells (2 × 105 ) were seeded on 24-well plates (Corning, Shanghai, China) and transfected with p65 or IKKβ expression plasmids by standard calcium phosphate precipitation. .. Luciferase assays were performed with a dual-specific luciferase assay kit (Promega, Durham, NC).

Article Title: Proteomic analysis of chicken embryo fibroblast cells infected with recombinant H5N1 avian influenza viruses with and without NS1 eIF4GI binding domain
Article Snippet: A firefly luciferase reporter plasmid pGL3-chIFN-β (100 ng/well) and a Renilla luciferase reporter plasmid pRL-TK (10 ng/well) were co-transfected with the indicated expression plasmids. .. At 24 h after transfection or further processing, dual-luciferase assays were performed using a dual-specific luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

Plasmid Preparation:

Article Title: The Us2 Gene Product of Herpes Simplex Virus 2 modulates NF-κB activation by targeting TAK1
Article Snippet: A Renilla luciferase reporter vector pRL-TK was used as an internal control. .. Luciferase assays were performed with a dual-specific luciferase assay kit (Promega Corp., Madison, WI, USA).

Article Title: TRIM32-TAX1BP1-dependent selective autophagic degradation of TRIF negatively regulates TLR3/4-mediated innate immune responses
Article Snippet: To normalize for transfection efficiency, pRL-TK (Renilla luciferase) reporter plasmid (0.01 μg) was added to each transfection. .. Luciferase assays were performed using a dual-specific luciferase assay kit (Promega, Madison, WI).

Article Title: PKACs attenuate innate antiviral response by phosphorylating VISA and priming it for MARCH5-mediated degradation
Article Snippet: To normalize for transfection efficiency, 0.01 μg of pRL-TK or pRL-SV40 (Renilla luciferase) reporter plasmid was added to each transfection [ , ]. .. Luciferase assays were performed using a dual-specific luciferase assay kit (Promega).

Article Title: WDFY1 mediates TLR3/4 signaling by recruiting TRIF
Article Snippet: Luciferase reporter assays were performed with a dual-specific luciferase assay kit (Promega). .. To normalize the transfection efficiency, 0.01 μg pRL-TK ( Renilla luciferase) reporter plasmid was added to each transfection.

Article Title: ZDHHC11 modulates innate immune response to DNA virus by mediating MITA–IRF3 association
Article Snippet: To normalize transfection discrepancies, 0.01 μg pRL-TK (Renilla luciferase) reporter plasmid was included in each transfection. .. Luciferase assays were performed using a dual-specific luciferase assay kit (Promega, Madison, WI, USA).

Article Title: Inducible Rubicon facilitates viral replication by antagonizing interferon production
Article Snippet: The Renilla luciferase reporter vector pRL-TK was used as an internal control. .. Luciferase assays were performed with a dual-specific luciferase assay kit (Promega, Madison, USA).

Article Title: USP13 negatively regulates antiviral responses by deubiquitinating STING
Article Snippet: Transfection and reporter gene assays HEK293 cells were transiently transfected with firefly luciferase reporter (100 ng) and TK-Renilla luciferase reporter (20 ng) and indicated plasmids or empty vector (100 ng) using standard calcium phosphate precipitation. .. After 24 h, luciferase assays were performed with a dual-specific luciferase reporter kit (Promega).

Article Title: TRIM27 mediates STAT3 activation at retromer-positive structures to promote colitis and colitis-associated carcinogenesis
Article Snippet: For normalization of transfection efficiency, 0.01 μg of pRL-TK (Renilla luciferase) reporter plasmid was added to each transfection. .. Luciferase assays were performed using a dual-specific luciferase assay kit (Promega).

Article Title: ANGPTL8 negatively regulates NF-κB activation by facilitating selective autophagic degradation of IKKγ
Article Snippet: To normalize transfection efficiency, 0.02 μg of pRL-TK Renilla luciferase reporter plasmid was added to each transfection. .. Luciferase assays were performed using a dual-specific luciferase assay kit (Promega), the firefly luciferase activities (NF-κB or IRF1 firefly luciferase reporter) were normalized based on Renilla luciferase activities.

Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway
Article Snippet: HEK293T cells (1 × 105 ) were seeded on 24-well and transfected with 100 ng of reporter plasmid, 20 ng of pRL-TK (Promega), and other indicated plasmids. .. At 24 hpt, the cells were mock infected or infected with SeV for 12 h; the luciferase activity was then measured by using a dual-specific luciferase assay kit (Promega).

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    Promega dual specific luciferase assay kit
    DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before <t>luciferase</t> assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a <t>dual-specific</t> luciferase <t>assay</t> <t>kit.</t> (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  
    Dual Specific Luciferase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dual specific luciferase assay kit/product/Promega
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    DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  

    Journal: Journal of Virology

    Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway

    doi: 10.1128/JVI.00588-19

    Figure Lengend Snippet: DNAJA3 restores VP1-induced inhibitory effect on IFN-β signaling pathway. (A) DNAJA3 restores the inhibition of VP1 on IFN-β signaling pathway in a dose-dependent manner. The HEK293T cells were transfected indicated plasmids and then mock infected or infected with SeV for 12 h before luciferase assays were performed. (B and C) VP1 inhibits the IFN-β or its downstream ISGs more efficiently on DNAJA3-KO PK-15 cells than on WT cells. DNAJA3-WT and DNAJA3-KO PK-15 cells were transfected with Flag-VP1 or vector. At 24 hpt, the cells were transfected by Lipofectamine 2000 with or without poly(I⋠C) at 50 ng/ml for 12 h. (B) Luciferase assays were performed using a dual-specific luciferase assay kit. (C) The IFN-β, ISG15, MX1, ISG54, and GBP1 mRNA levels were detected by RT-PCR. **, P  

    Article Snippet: At 24 hpt, the cells were mock infected or infected with SeV for 12 h; the luciferase activity was then measured by using a dual-specific luciferase assay kit (Promega).

    Techniques: Inhibition, Transfection, Infection, Luciferase, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.

    Journal: Journal of Virology

    Article Title: Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway

    doi: 10.1128/JVI.00588-19

    Figure Lengend Snippet: FMDV VP1 protein inhibits the SeV-induced IFN-β signaling pathway. (A) The expression of VP1 inhibits SeV-induced activation of IFN-β promoters. HEK293T cells were transfected with Flag-VP1 or empty vector, together with IFN-β luciferase reporter. At 24 hpt, the cells were mock infected or infected with SeV for 12 h before luciferase assays were performed using a dual-specific luciferase assay kit. (B) VP1 negatively regulates SeV-induced activation of IFN-β at the IRF3 level. HEK293T cells were transfected with IFN-β reporter, pRL-TK, expression plasmids for Flag-VP1, and the indicated protein. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (C, D, and E) FMDV VP1 inhibits the phosphorylation, dimerization, and nuclear translocation of IRF3 after SeV stimulation. HEK293T cells were transfected with the indicated plasmids for 24 h. Cells were infected with SeV at various time points and then harvested for analysis by Western blotting (C) or native-PAGE (D). (E) Cells were stained with the indicated antibodies and imaged by confocal microscopy.

    Article Snippet: At 24 hpt, the cells were mock infected or infected with SeV for 12 h; the luciferase activity was then measured by using a dual-specific luciferase assay kit (Promega).

    Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Infection, Western Blot, Translocation Assay, Clear Native PAGE, Staining, Confocal Microscopy