To explore the effect of SEPT9_v2 on T cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activities, TOPflash luciferase reporter plasmids containing TCF/LEF binding sites and FOPflash luciferase reporter plasmids containing a mutant TCF/LEF binding site were assayed. Cells were seeded into 24-well plates and were transiently co-transfected with TOPflash or FOPflash luciferase reporter plasmids and SEPT9_v2 or vector (pcDNA3.1). Renilla luciferase reporter pRL-TK (Promega) was added to each well as an internal control. After 48 h, a dual-luciferase reporter assay kit (Promega) was used to detect luciferase activity according to the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity and presented as a relative activity. To verify FZD10 as a direct target of miR-92b-3p, luciferase reporter plasmids (Guangzhou Ribobio Co, Ltd, Guangzhou, China) containing WT or MT FZD10 3′-UTR (3′-untranslated region) were assessed. Cells were seeded into 24-well plates and were transiently co-transfected with WT or MT FZD10 3′-UTR reporter plasmids and SEPT9_v2 or vector (pcDNA3.1). Renilla luciferase reporter pRL-TK (Promega) was added to each well as an internal control. The follow-up steps were performed as described above.
Renilla luciferase reporter prl tk
Manufactured by Promega
Sourced in United States
The pRL-TK Renilla luciferase reporter vector is a control reporter plasmid used in co-transfection experiments to normalize for transfection efficiency. The vector expresses the Renilla luciferase gene under the control of the herpes simplex virus thymidine kinase (HSV-TK) promoter.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (9 mentions)
Dual luciferase reporter assay kit, by Promega (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Glomax 20 20 luminometer, by Promega (3 mentions)
Passive lysis buffer (plb), by Promega (3 mentions)
Pegfp c1, by Takara Bio (2 mentions)
Polyjet in vitro dna transfection reagent, by SignaGen (2 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (2 mentions)
Pgl3 luciferase reporter vector, by Promega (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Promega (2 mentions)
Isre luc, by Takara Bio (1 mentions)
Pegfp c1, by Addgene (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Fopflash reporter, by Merck Group (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Yy1 sirna, by Horizon Discovery (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Dual luciferase reporter assay system kit, by Promega (1 mentions)
Luciferase reporter plasmid, by RiboBio (1 mentions)
26 protocols using renilla luciferase reporter prl tk
1
Investigating SEPT9_v2 Regulation of TCF/LEF Transcription
2
Recombinant SARS-CoV-2 Viral Proteins Production
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Gene fragments of all 27 viral proteins (except PLpro of SARS-CoV-2) were gene synthesized (Sangon Biotech) with codon optimization. PLpro and orf6 of both SARS-CoV and SARS-CoV-2 were PCR amplified from cDNA reverse-transcribed from viral RNA extracted from viral supernatant. All gene fragments were cloned into pCAGEN expression vector with c-terminal FLAG-tag and confirmed by Sanger sequencing. Plasmids used have previously been detailed [33 (link)]. pEBG plasmid containing the GST-tagged RIG-I N-terminal card domains was used. Interferon-beta firefly luciferase reporter (IFNβ-luc), which comprises the promoter of IFNβ, is a kind gift of Professor Takashi Fujita, Kyoto University. Interferon-stimulated response element firefly luciferase reporter (ISRE-luc) which contains five repeats of ISRE enhancer element upstream of minimal TA promoter was purchased from Clontech. Renilla luciferase reporter (pRL-TK) that contains an HSV-thymidine kinase promoter was purchased from Promega.
3
Plasmid Vectors for Protein Expression and Transcription Assays
4
Plasmid Purification and Transfection
5
Transcription Factor Binding Site Identification
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The transcription factor binding sites (TFBS) were identified by Genomatix. pGL4.12-Luc vector (Promega) including the indicated cloned genomic regions and thymidine kinase (TK) promoter was transfected into 293T cells. A Renilla luciferase reporter pRL-TK (Promega) was co-transfected as a control for evaluating transfection efficiency. Transfections were performed with Lipofectamine 2000 or LTX (Invitrogen) according to the manufacturer's instructions. After 24 h, cells were washed before measuring firefly and Renilla luciferase activities using the Dual-Glo™ Luciferase Assay System (Promega). In each experiment, firefly luciferase activity was normalized to Renilla luciferase activity.
6
Dual-Luciferase Reporter Assay in RGCs
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TOPFlash reporter or FOPFlash reporter (Millipore, Billerica, MA, USA) was co-transfected with an internal control Renilla luciferase reporter pRL-TK (Promega, Madison, WI, USA) into primary RGCs with the Micropoly-transfecterTM cell reagent. Twenty-four hours after transfection, cells were subjected to the respective treatment for another 24 h. Cells were harvested and analyzed by the dual-luciferase assay kit (Promega, Madison, WI). Three independent experiments were performed in duplicate for each condition.
7
MERVL-luciferase Reporter Assay in ESCs
8
MERVL-luciferase Reporter Assay in ESCs
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For MERVL-luciferase reporter assays, we used the pGL3 luciferase reporter vectors (Promega, Cat. # E1751) that harbors the MERVL125–375-fragment previously described (MERVL-Luc) (Choi et al., 2017 ). MERVL-Luc reporters and control Renilla luciferase reporter pRL-TK (Promega, Cat. # E2241) were co-transfected into ESCs (600 ng and 150 ng per well of a 12-well plate, respectively), using Lipofectamine 2000 (Life Technologies, Cat. # 11668027). Transfection complexes containing the reporter constructs were prepared in Opti-MEM Reduced-Serum Medium (Life Technologies, Cat. # 31985062). After trypsinization with 0.25% Trypsin + EDTA (Life Technologies, Cat. # 25200–056), 100,000 cells were resuspended in ES media lacking Pen Strep, incubated with transfection complexes for 10 minutes at 37°C, and then transferred to one well of a 12-well plate containing irradiated MEF feeders. After 48 hours, transfected ESCs were trypsinized, plated onto gelatin-coated plates for 1 hour to remove feeders, and then assayed for luciferase activity by Dual-Luciferase Reporter Assay System (Promega, Cat. # E1910) using a Glomax 20/20 Luminometer (Promega).
9
Cloning and Transfection of pri-miR-181a/b1 Enhancer
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The pri-miR-181a/b1 enhancer region was cloned into the pGL4.27 Luc2p/minP vector (Promega) as described28 (link). Thirty nanograms of pri-miR-181a/b1 Luc2p/Peak 1 was co-transfected with 1 ng Renilla luciferase reporter pRL-TK (Promega) into HEK293T cells, or Luc2p empty vector (negative control). For TCF1 loss-of-function on pri-miR-181a/b1 enhancer activity, TCF7 siRNA (Dharmacon A-019735-13-0005) or non-targeting siRNA (Dharmacon D-001910-01-05) were co-transfected; YY1 siRNA (Dharmacon A-011796-16-0005) was transfected as a positive control. For gain-of-function, different amounts of pCDNA3-HA-TCF7 (0, 30 or 100 ng, Addgene #40620) or pCDNA3-CTNNB1 plasmids (0, 30, 100 ng, Addgene #16828) were co-transfected. As for GSK3β inhibitor treatments, 1 µM BIO or 10 µM SB216763 was added 12 h after transfection. Cells were collected 48 h later, and enhancer activity was determined using the Dual Luciferase Reporter Assay System (Promega, E1910) as described by the manufacturer’s protocol.
10
Evaluation of NF-κB Reporter Activity
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LNCaP and 22Rv1 cells (density 5 × 104/mL) were seeded in 24-well plates (n = 6). After 24 h, the cells in each well were transfected with a mixture of 1.0 µL FuGENE® HD Transfection Reagent (Promega, Madison, WI, USA), 0.2 µg NF-κB reporter plasmid pNF-κB-TA-Luc (Takara Bio Inc., Shiga, Japan), 0.04 µg Renilla luciferase reporter pRL-tk (Promega), and 0.1 µg pEGFP, 0.1 µg pEGFP-AR-FL, or 0.1 µg pEGFP-AR-V7. The NF-κB reporter activities were measured 24 h after transfection using the Dual-Luciferase® Reporter Assay System (Promega). Both the firefly and Renilla luciferase activities were measured according to the manufacturer’s instructions. The cells were washed three times with phosphate-buffered saline. Passive lysis buffer was added to each well and was shaken at room temperature for 15 min. Lysate (20 µL) from each sample was transferred to 96-well plate for firefly and Renilla luciferase activity measurements by a luminometer [20 (link)].
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