The Dulbecco modified Eagle medium (DMEM), optiMEM, trypsin (0.25%), L-glutamine, penicillin, streptomycin, fetal bovine serum (FBS), and the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and PI were purchased from Gibco (Gaithersburg, MD, USA). The andrographolide (≥98% purity), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), SB203580, ethylenediaminetetraacetic acid (EDTA), and sodium stibogluconate (SSG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The p38MAPK inhibitor III was purchased from Millipore (Billerica, MA, USA). The Turbofect in vitro transfection reagent was purchased from Upstate Biotechnology (Lake Placid, NY, USA). The PG13-luc plasmid (Addgene, Cambridge, MA, USA) containing the p53 binding site was kindly provided by Dr. Bert Vogelstein36 (link). The Renilla-luc and Dual-Glo luciferase assay systems were purchased from Promega (Madison, WI, USA). The 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine chloride (JC-1) was purchased from BioVision (Mountain View, CA, USA). The Hybond-P polyvinylidene difluoride (PVDF) membrane, enhanced chemiluminescence (ECL) detection reagent were purchased from Amersham (Buckinghamshire, UK). andrographolide was dissolved in 0.1% dimethyl sulfoxide (DMSO), and stored at 4°C.
Pg13 luc plasmid
Manufactured by Addgene
Sourced in United States
The PG13-luc plasmid is a reporter construct that contains the luciferase gene under the control of the PG13 promoter. This plasmid is commonly used for the study of p53-dependent transcriptional regulation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Sb203580, by Merck Group (1 mentions)
Andrographolide, by Merck Group (1 mentions)
Dual glo luciferase assay, by Promega (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
Trypsin 0.25, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay kit, by Promega (1 mentions)
Synergy h1 plate, by Agilent Technologies (1 mentions)
Prl tk plasmid, by Promega (1 mentions)
2 protocols using pg13 luc plasmid
1
Andrographolide Induces p53-Dependent Apoptosis
2
Evaluating Axin and P53 Peptide Analogues
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For evaluation of Axin peptide analogues, DLD-1 cells were cultured in 96-well flat bottom plates up to 70–80% confluency and were transfected with 200 ng M50 TOPFlash plasmid (a gift from Professor Raymond Habas, Temple Biology) and 50 ng pRL-TK plasmid (Promega). 12 h after transfection, cells were treated with DMSO vehicle or 15 μM of Axin peptide analogues for another 24 h. Luciferase activity was measured using the Dual-Glo luciferase assay kit (Promega) on a Biotek Synergy H1 plate reader using the Gen5 software. Data were processed as firefly: Renilla luciferase ratio for each control or treatment group, and were further normalized against the vehicle control group (100%) as the Relative Activities.
For evaluation of P53 peptide analogues, HCT116 wt cells were transfected with 200 ng PG13-luc plasmid (a gift from Bert Vogelstein (Addgene plasmid #16442;http://n2t.net/addgene:16442 ; RRID:Addgene_16442)) and 50 ng pRL-TK plasmid. At about 12 h after transfection, the cells were treated with solvent vehicle or 15 μM of each p53 peptide analogue for an additional 24 h. The follow-up luciferase activity measurement and data processing were completed in the same manner as mentioned above.
For evaluation of P53 peptide analogues, HCT116 wt cells were transfected with 200 ng PG13-luc plasmid (a gift from Bert Vogelstein (Addgene plasmid #16442;
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