6 × 105 cells ml−1 ΔFZD1-10 HEK293 T cells were seeded onto PDL-coated white 96-well cell culture plate with solid flat bottom (Greiner Bio-One). Next day, cells were transfected with 20 ng of SNAP-tagged receptor, 20 ng M50 Super 8× TOPFlash (Addgene, 12456), 2 ng pRL-TK Luc (Promega, E2241) and 58 ng pcDNA plasmid DNA to a final amount of 100 ng of plasmid DNA per well. Four hours after transfection, medium was changed to starvation medium (DMEM without FBS) containing either vehicle or 1000 ng/ml recombinant WNT-3A for FZD transfected cells or 100 nM SAG1.3 for SMO-transfected cells. Twenty-four hours after stimulation, cells were lysed gently shaking with 20 µl 1× Passive Lysis Buffer (Promega, E1910) for 15 min. Subsequently, 20 µl of LAR II (Promega, E1910) were added to all wells after which luminescence (580-80 nm) was read and then 20 µl of Stop & Glo (Promega, E1910) were added to all wells after which luminescence (480-80 nm) was read again with a CLARIOstar microplate reader (BMG). Data were analyzed using GraphPad Prism 6.
Prl tk luc
Manufactured by Promega
Sourced in United States
The PRL-TK-luc is a reporter construct that contains the luciferase gene under the control of the prolactin (PRL) promoter. It is designed to measure the transcriptional activity of the PRL promoter in cells.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase assay kit, by Promega (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Dual luciferase reporter gene assay, by Promega (3 mentions)
M50 super 8 topflash, by Addgene (2 mentions)
Lar 2, by Promega (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
M50 super 8x topflash, by Addgene (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Glo lysis buffer, by Promega (2 mentions)
Dual luciferase assay system, by Promega (2 mentions)
Stop glo, by Promega (1 mentions)
Pdl coated white 96 well cell culture plate with solid flat bottom, by Greiner (1 mentions)
Super8x topflash, by Promega (1 mentions)
Pegfp c2, by Takara Bio (1 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Lipofectamine ltx with plus reagent, by Thermo Fisher Scientific (1 mentions)
Synergy 2 microplate reader, by Agilent Technologies (1 mentions)
Pgl4.22 nf κb, by Promega (1 mentions)
Pgl4.22 vector, by Promega (1 mentions)
Firefly luciferase plasmid, by Promega (1 mentions)
17 protocols using prl tk luc
1
Wnt Signaling Pathway Activation Assay
2
Plasmid constructs for DVL study
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The plasmids used were described previously and included pcDNA3-Flag-mDVL1; DVL-pcDNA3-Flag-mDVL1 (aa 1 to 502), pcDNA3-Flag-mDVL1 (DVL1 aa 1 to 394), pcDNA3-Flag-mDVL1 (DVL1 aa 1 to 345), pcDNA3-Flag-mDVL1 (DVL1 aa 1 to 250), pcDNA3-Flag-mDVL1 (DVL1 aa 1 to 217) (17 (link)) carrying DVL1 with truncations or point mutations; pCDNA3.1-(zeo)-Venus-hDVL1 and pCDNA3.1-Flag-hDVL3 (34 (link)); pcDNA3.1-HA-mDVL2 (7 (link)); pEGFP-C2 from Clontech; pRLtkLuc from Promega; Super8X TopFlash (35 (link)); pEGFP-C1-mDVL2 and pEGFP-C1-hDVL3 (36 (link)); pCMV5-3×-FLAG-hDVL2 (37 (link)); and pCMV hDvl3-HA (38 (link)). Site-directed mutagenesis of pcDNA3-Flag-mDVL1(1–502) was performed by use of a QuikChange II XL site-directed mutagenesis kit (Agilent) according to the manufacturer's instructions. The sequences of all mutants described in this study were verified by sequencing.
3
Measuring GLI-mediated Transcriptional Repression
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To measure the GLI‐mediated transcriptional repression in response to cyclopamine, B16F10 cells were plated in 12‐well plates at 8 × 104 cells per well, and were cotransfected with 950 ng of the Luc reporter vector (8xGLI‐BS‐Luc or 8xmGli‐BS‐Luc, kind gifts from Dr Hiroshi Sasaki, Kumamoto University, Kumamoto, Japan)37 and 50 ng of the Renilla Luc control vector (pRL‐TK‐Luc, Promega) using Lipofectamine LTX with Plus reagent (Thermo Fisher Scientific). Six hours after transfection, the medium was changed, and the cells were treated with either vehicle (DMSO) or cyclopamine for 24 h. The cells were then lysed, and the Luc activity was measured as previously described.34 To examine the effect of GLI1 overexpression on the potential promoters of Snail1, Zeb1 and Twist1, 350 ng of the Luc reporter vector containing the potential promoter region, 140 ng of either pCL20c‐CMV‐EGFP or pCL20c‐CMV‐HA‐GLI1, and 10 ng of pRL‐TK‐Luc were cotransfected into HEK293T cells plated in 12‐well plates at 2 × 105 cells per well. Thirty‐six hours after transfection, the cells were lysed, and subjected to Luc assays as described previously.34
4
Wnt Signaling Activation Assay in HEK293
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HEK293ΔFZD1-10 cells55 were seeded onto 48-well plates and the next day cells were transfected with M50 Super 8x TOPFlash (Addgene #12456), pRL-TK Luc (Promega E2241), FZD5 and empty vector. 4 h post transfection, medium was changed to starvation medium with or without WNT-3A (300 ng ml−1; Biotechne 5036-WN). 24 h after transfection, cells were analyzed by the Dual-Luciferase Reporter Assay System (Promega E1910) according to manufacturer’s instructions in white 96-well plates with the following modifications: cells were lysed in 50 µl Passive Lysis Buffer, Stop & Glo reagent was used at 0.5X and 25 µl of LARII and Stop & Glo Reagent were used for each well. Luminescence was measured using a Synergy2 microplate reader (BioTek).
5
NF-κB Promoter-Driven Luciferase Assay
6
TGF-β Signaling Modulation via BGN siRNA
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For reporter gene assays, Panc1 cells were seeded in 96-well plates (NunclonTM Delta Surface) and cotransfected on the following day serum-free for 4 h with Lipofectamine 2000 (Life Technologies) and 50 nM of BGN siRNA or control siRNA. Afterwards, cells received standard growth medium. Twenty-four h after the start of the first transfection, cells underwent a second round of transfection with 50 nM each of BGN or control siRNA plus 100 ng/well of the TGF-β-responsive luciferase reporter plasmid p3TP-Lux and 25 ng/ml pRL-TK-Luc, a vector encoding Renilla luciferase (Promega, Heidelberg, Germany). On the next day, cells were stimulated with 5 ng/ml TGF-β1 for 24 h and then lysed in Glo lysis buffer (Promega) and subjected to dual luciferase measurement with the Dual Luciferase Assay System according to the manufacturer’s protocol (Promega).
7
Dual Luciferase Assay in HEK293 Cells
8
Dual-Luciferase Reporter Assay for AP-1 and NF-κB
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HEK-293 or MCF-7 cells were transfected with pGL4.22-AP1 (gift from Dr. D. Sanchez) or pGL4.22-NF-κB (Promega, Madison, WI, USA), mixed with pRL-TK-luc (Promega) at a 40:1 ratio using FuGENE HD Transfection Reagent (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. Twenty-four hours after transfection, the cells were exposed to test agents for another 24 h (for AP-1) or 5 h (for NF-κB). Cell lysates were used for determining luciferase activities of both firefly and renilla by the dual luciferase reporter gene assay (Promega). Firefly luciferase activity was normalized to renilla luciferase activity. The experiment was carried out in triplicate and expressed as the mean ± SD.
9
ARE-Driven Luciferase Assay for Nrf2 Activity
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The luciferase reporter construct pGL4.22-ARE was a gift from Dr. Donna Zhang at University of Arizona. It was generated by cloning a 39-bp ARE-containing sequence from the promoter region of the NAD(P)H quinone oxidoreductase 1 (NQO1) into the pGL4.22 vector (Promega, Madison, WI) [41 (link)]. The MCF-10A or MCF-7 cells were transfected with the pGL4.22-ARE plasmid and a constitutively active renilla luciferase (pRL-TK-luc, from Promega; to correct for tranfection efficiency) (40:1 ratio) using FuGENE HD Transfection Reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s instructions. Twenty-four hours after transfection, the cells were exposed to the extracts of SWT or components for another 24 hours. Cell lysates were used for determining luciferase activities of both firefly and renilla by the dual luciferase reporter gene assay (Promega). Firefly luciferase activity was normalized to renilla luciferase activity. The experiment was carried out in triplicate and expressed as the mean ± SD.
10
Investigating NF-κB and AP-1 Regulation
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HEK293T cells were plated in 96-well plates and transfected with plasmids carrying an NF-κB or AP-1 luciferase reporter (firefly luciferase plasmid, Promega, Madison, WI, USA) and pRL-TK-luc (Renilla luciferase plasmid, Promega) together with plasmids expressing ANXA1 or the SUMOylation mutants of ANXA1. OGD/R treatment or MyD88, TRAF2, TRAF6, RIP1, IKKα, IKKβ, IKKγ, IκBα shRNA, or p65 was used as stimulators. Cells were lysed at 24 hours after transfection, and luciferase activity was detected with the Dual-Luciferase Assay kit in accordance with the manufacturer’s instructions (Promega) using a Fluoroskan Ascent FL system (Thermo Fisher Scientific). The reporter gene activity was examined by normalizing the firefly luciferase activity to Renilla luciferase activity. The experiments were performed in triplicate.
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