Plasmid pSTAT3-TA-luc (Beyotime Biotechnology, Shanghai, China) contains multiple copies of the STAT3-binding site at its polyclonal site and its activation specifically depends on STAT3 status in the cell environment. pSTAT3-TA-luc plasmids were transfected into HeLa cell lines using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The stable clones, which showed high luciferase activity, were seeded into 96-well plates and cultured with or without TCN for 4 h, then, luciferase activity was measured by using a Steady-Glo® Luciferase Assay System (Promega, Madison, WI, USA), following the manufacturer’s instructions. The cell viability was detected by SRB assay.
Pstat3 ta luc
Manufactured by Beyotime
Sourced in China
The PSTAT3-TA-luc is a luciferase reporter assay system designed to measure the transcriptional activity of the STAT3 (Signal Transducer and Activator of Transcription 3) transcription factor. It contains a STAT3-responsive luciferase reporter construct that can be used to quantify STAT3 activation in cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Steady glo luciferase assay system, by Promega (1 mentions)
Dual luciferase assay protocol, by Promega (1 mentions)
Prl tk, by Promega (1 mentions)
Control sirna sictrl, by Santa Cruz Biotechnology (1 mentions)
Dual luciferase reporter assay system, by Beyotime (1 mentions)
Lux multimode microplate reader, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Prl tk, by Beyotime (1 mentions)
Beyogold 96 well white opaque plates, by Beyotime (1 mentions)
Pnfκb luc, by Beyotime (1 mentions)
Prl tk plasmid, by Beyotime (1 mentions)
Prl tk plasmid, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Prl tk renilla, by Beyotime (1 mentions)
Dual luciferase reporter kit, by Beyotime (1 mentions)
7 protocols using pstat3 ta luc
1
Evaluation of STAT3-dependent Luciferase Activity
2
STAT3 Transcriptional Activity Assay
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HeLa cells were co-transfected with pSTAT3-TA-luc (Beyotime) and pRL-TK (Promega) as a transfection efficiency control in a 6-well plate. After 12 h, cells were transferred to a 48-well plate with complete medium and cultured for 24 h. Cells were pre-treated with different concentration of compounds (in 0.05% DMSO) for 6 h in low FBS medium, and EGF (100 ng/ml) was subsequently added into the wells for 30 min to stimulate STAT3 expression and the cells were incubated for 30 min. In the IFN-α-induced comparative assay, HeLa cells were co-transfected with p-GAS-TA-luc (Biovector, Beijing, China) and pRL-TK in a 24-well plate for 6 h. Cell were cultured with complete medium for another 18 h. The indicated concentrations of compound 1 and S3I-201 were added to each well for 5 h, followed by the addition of 1000 U/ml IFN-α for 1 h to stimulate STAT3/STAT1 expression. Cell lysates were collected according to the dual luciferase assay protocol (Promega). Sample light output was analyzed using a luminometer and the resulting data were normalized relative to pRL-TK values.
3
Adenovirus-Mediated GRIM-19 Overexpression and Silencing
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Generation and transfection of recombinant adenovirus-GRIM-19 (Ad-GR19) and GFP control (Ad-GFP) were performed as described previously [21 (link)]. A pooled GRIM-19 shRNA (shGR19) plasmids (Santa Cruz Biotechnology, Santa Cruz, CA) was used to knockdown GRIM-19 expression in indicated cells and a scramble shRNA (shCON) was used as control. ShRNA plasmids were transfected into indicated cells and resistant colonies were selected with puromycin (1ug/ml). Individual clones were isolated and expanded in the selection medium. Pooled STAT3 siRNA (siSTAT3) and Control siRNA (siCtrl) were purchased from Santa Cruz Biotechnology. STAT3 luciferase reporter plasmid pSTAT3-TA-luc was purchased from Beyotime Inc (Haimen, China). GRIM-19-GFP recombinant was generated by inserting GRIM-19 cDNA to the GFP upstream of pEGFP-N1 plasmids (Clontech) [21 (link)]. Transfection of plasmids or siRNAs was performed using FuGENE HD transfection reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions.
4
Dual-Luciferase Reporting in NPC Cells
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The pNFκB-luc, pSTAT3-TA-luc, and pRL-TK plasmids were purchased from Beyotime. NPC cells were seeded in BeyoGold™ 96-Well White Opaque Plates (Beyotime) transfected with Lipofectamine™ 3000 Reagent (Invitrogen, Carlsbad, CA, USA). Reporter enzyme activity was determined with a dual-luciferase reporter assay system (Beyotime) according to the manufacturer’s instructions. The luminescence signal was determined with a Varioskan LUX multimode microplate reader (Thermo). Relative luminescence units = Firefly luciferase activity/Renilla luciferase activity.
5
Quantifying STAT3 Transcriptional Activity
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5×105cells were plated into 60 mm culture dish and incubated with RPMI-1640 medium containing 10% FBS for 12h. Cells were transiently cotransfected with 0.5 µg of a reporter plasmid containing human STAT3 response element (pSTAT3-TA-luc) (Beyotime, Nanjin, China) and 0.5 µg of pRL-TK plasmid (Promega, Madison, WI, USA) using lipofectamine 2000 in serum-free medium. Cotransfection of pGL6-TA without STAT3 response element (Beyotime) and pRL-TK plasmid into cells served as a control. Cells were harvested 48h after transfection, and both firefly luciferase and renilla luciferase activities were measured with the Dual-luciferase reporter assay system (Promega) according to the manufacturer’s instruction, and transcriptional activity of Stat3 was estimated using a luminometer.
6
AMPK Modulators and Luciferase Reporters
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Dominant negative AMPKα1 plasmid, pDN-AMPKa1 (T172A) was a gift from Dr. Jae Bum Kim; constitutive activated AMPKα1 plasmid, pCA-AMPKα1 (T172D) encodes AA1–312 of AMPKα1 with threonine 172 mutated to aspartic acid was created. Firefly luciferase expressing plasmid with STAT3 promoter (pSTAT3-TA-luc, Cat: D2259) was from Beyotime Biotechnology. pSHP-luc was established by amplifying the mouse SHP promoter (-2K) and inserting it into PGL3-Basic Vector. Endoxin-free plasmids were transfected using Jet PRIME transfection reagent following manufacturer's instruction.
7
Shikonin Modulates STAT3 Transcriptional Activity
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Lung adenocarcinoma cells (A549 and H1299) were seeded in 48-well plates at the number of 3×104 cells/well and cultured under normal conditions for 24 h, then transfected with 0.2 µg pSTAT3-TA-luc (Beyotime Institute of Biotechnology) and 0.01 µg pRL-TK Renilla (Beyotime Institute of Biotechnology) using Lipofectamine 2000. Twenty-four hours after transfection, the cells were treated with shikonin and IL-6 (50 ng/ml) for 24 h. After treatment, the cells were lysed using passive lysis buffer, and the luciferase activity was detected using a Dual Luciferase Reporter kit (Beyotime Institute of Biotechnology). The experiment was carried out in triplicate and repeated three times independently.
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