HEK293 cells were transfected with pU6-puro-siTAF-I or pU6-puro-siEGFP together with pISRE-TA-Luc (Clontech) containing the ISRE and pSEAP-Control (Clontech) using Gene Juice (Novagen) according to the manufacturer's protocol. At 48 h post transfection, cells were treated with or without IFN-β (1000 IU/ml) for 6 h. Cell lysates were used for assays of the luciferase activity using the luciferase assay system (Promega) and a MiniLumat LB9506 luminometer (Berthhold). To monitor the transfection efficiency, a portion of each cell culture medium was assayed for secreted alkaline phosphate (SEAP) using the SEAP assay kit (Toyobo).
Pisre ta luc
Manufactured by Takara Bio
Sourced in United States
PISRE-TA-Luc is a laboratory equipment product from Takara Bio. It functions as a reporter assay system for the analysis of transcriptional activation.
Automatically generated - may contain errors
Lab products found in correlation
Pta luc, by Takara Bio (2 mentions)
Pp53 ta luc, by Takara Bio (2 mentions)
Luciferase assay system, by Promega (1 mentions)
Prl cmv, by Promega (1 mentions)
Pmscv puro retroviral vector, by Takara Bio (1 mentions)
Pcre luc, by Takara Bio (1 mentions)
Phse luc, by Takara Bio (1 mentions)
Pe2f ta luc, by Takara Bio (1 mentions)
Pap1 luc, by Takara Bio (1 mentions)
Pnf κb ta luc, by Takara Bio (1 mentions)
Psre luc, by Takara Bio (1 mentions)
Pstat3 ta luc, by Takara Bio (1 mentions)
Fopflash, by Addgene (1 mentions)
Topflash, by Addgene (1 mentions)
Pmirglo vector, by Promega (1 mentions)
Pap1 pma ta luc, by Takara Bio (1 mentions)
Victor x5 multilabel plate reader, by PerkinElmer (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Prl tk plasmid, by Promega (1 mentions)
5 protocols using pisre ta luc
1
Quantifying IFN-β-Induced Transcriptional Activity
2
Luciferase Assay for IRF9 Translocation
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A luciferase reporter assay was performed to assess the effects of TMEM2-induced IRF9 translocation into nuclei. Plasmid pISRE-TA-Luc (Clontech) containing five copies of consensus ISRE upstream of the firefly luciferase gene was used for cell transfection. Plasmid pTA-Luc (Clontech) lacking the enhancer element was used for background determination. Plasmid pRL-CMV (Promega) expressing the Renilla luciferase protein was used to normalize the transfection efficiency. To measure IRF9-induced ISRE activities, 5 × 104 cells per well were seeded in a 24-well plate 1 day before transfection. Five hundred nanograms of pISRE-TA-Luc or pTA-Luc with 1 ng of pRL-CMV were used for cell transfection with 2 μl of Lipofectamine 2000 according to the manufacturer's instructions. Forty-eight hours after transfection, dual luciferase assays were performed using the cell lysates according to established protocols.
3
Cloning and Profiling of miR-424-5p
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The human miR-424-5p gene was PCR amplified from genomic DNA and cloned into a pMSCV-puro retroviral vector (Clontech, Japan). Pathway Profiling System, including pAP1-luc, pCRE-luc, pE2F-TA-luc, pERE-luc, pGRE-luc, pHSE-luc, pISRE-TA-luc, pMYC-luc, pNF-κB-TA-luc, pp53-TA-luc, pSRE-luc, and pSTAT3-TA-luc, was purchased from Clontech (PT3286-1). HOPFlash (Catalog [Cat.] 83467), HIPFlash (Cat. 83466), TOPFlash (Cat. 12456), and FOPFlash (Cat. 12457) were purchased from Addgene. The 3′ UTRs of WWC1, SAV1, and LATS2 were PCR amplified from genomic DNA and cloned into pmirGLO vectors (Promega, USA), and the list of primers used in cloning reactions was provided in Table S3 . Anti-miR-424-5p was synthesized and purified by GENECHEM (China). The primers of the mutant plasmid for WWC1, SAV1, and LAST2 were synthesized and purified by HDbio (Guangzhou, China). Transfection of plasmids was performed as previously described.46 (link)
4
Pathway Reporter Assays in ESCs
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Pathway reporter vectors pAP1-TA-luc, pAP1 (PMA)-TA-luc, pISRE-TA-luc, pP53-TA-luc, and the negative control pTA-luc were purchased from Clontech Laboratories, Inc. (Mountain View, CA, USA). Other signaling transduction reporter vectors including pCRE-TA-luc and pGRE-TA-luc were constructed in our laboratory by inserting their cis-acting DNA binding sequence into the multiple cloning sites of pTA-luc [31 (link)]. Luciferase assays were performed with the dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions. Briefly, pathway reporter vectors and pRL-SV40 were cotransfected into ESCs by Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. At 24 h after transfection, 1 μM PD or an equal volume of DMSO was added to culture medium for another 24 h. Cells were then lysed in passive lysis buffer and luciferase activity was measured on a VICTOR X5 Multilabel Plate Reader (PerkinElmer, Norwalk, CT, USA).
5
ISRE-Mediated Signaling Pathway Monitoring
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The pISRE-TA-Luc (Clontech) reporter plasmid was used for monitoring the induction of ISRE-mediated signal transduction pathways. The cells (5 × 104) grown in 48-well plates were transfected with 0.4 μg/well of a reporter plasmid, 0.4 μg/well of an empty plasmid or 0.2 μg/well of a reporter plasmid and 0.2 μg/well of expression plasmid, along with 0.1 μg/well of pRL-TK plasmid (Promega). Cells were infected with reovirus and Sendai virus (SeV, positive control) 12 h after transfection. Luciferase assays were performed 12 h after SeV infection and at indicated time points after reovirus infection. Luciferase activity in these cultures was quantified using the Dual-Luciferase Assay kit (Promega) according to the manufacturer’s instructions.
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