Assay for MEK4-Mediated Transcriptional Regulation

Constructs were purchased or gifts: constitutive active MEK4EE (MAP2K4-EE; residues 37-399; Addgene, plasmid 14813), pRL-TK-Renilla luciferase (Promega), pCMV-β-galactosidase (Agilent Technologies), and pcDNA-GFP (Invitrogen), HSP90β was from Pawel Bieganowski (Mossakowski Medical Research Center PAS, Poland)^{34 (link)}, estrogen-responsive promoter-luciferase reporter construct, pERE-Luc, was from Craig Jordan (Georgetown University)^{35 (link)}, human CDC37 in pET15b plasmid was from Avrom Caplan (City College of New York)^{36 (link)}. siRNA used Dharmacon ON-Targetplus SMARTpool™ siRNA directed against HSP90β (cat # L-005187-00-0010) non-targeting siRNA (cat # D-001810-10-05) used TransIT-LT1 Transfection Reagent (Mirus Bio LLC), or with Dharmafect Duo (Thermo Scientific, Lafayette, CO) for co-delivery of plasmid. Luciferase assays were performed as described by us^{30 (link)}.

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Xu L., Gordon R., Farmer R., Pattanayak A., Binkowski A., Huang X., Avram M., Krishna S., Voll E., Pavese J., Chavez J., Bruce J., Mazar A., Nibbs A., Anderson W., Li L., Jovanovic B., Pruell S., Valsecchi M., Francia G., Betori R., Scheidt K, & Bergan R. (2018). Precision therapeutic targeting of human cancer cell motility. Nature Communications, 9, 2454.

Publication 2018

pRL-TK-Renilla luciferase ON-Targetplus SMARTpool™ TransIT-LT1 Transfection Reagent Dharmafect Duo

Assays Delivery Estrogen Gifts Human Luciferase Map2k4 Plasmid Promega Renilla luciferase Sirna Transfection β galactosidase

Corresponding Organization :

Other organizations : Northwestern University, Oregon Health & Science University, University of Chicago, University of Washington, The University of Texas at El Paso

Top 5 similar protocols

Variable analysis

independent variables

Constitutive active MEK4EE (MAP2K4-EE; residues 37-399)
SiRNA directed against HSP90β
Non-targeting siRNA

dependent variables

Luciferase activity

control variables

PRL-TK-Renilla luciferase
PCMV-β-galactosidase
PcDNA-GFP
HSP90β
Estrogen-responsive promoter-luciferase reporter construct, pERE-Luc
Human CDC37 in pET15b plasmid

positive controls

Constitutive active MEK4EE (MAP2K4-EE; residues 37-399)
Estrogen-responsive promoter-luciferase reporter construct, pERE-Luc

negative controls

Non-targeting siRNA

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