PMC:2783367 / 9650-11962 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/2783367","sourcedb":"PMC","sourceid":"2783367","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2783367","text":"Plasmids and transient transfection assays\nThe lox-1 promoter luciferase reporter plasmid (p-2336/+36-Luc), containing a 2336 bp fragment of the 5′-flanking promoter region of the LOX-1 gene, and some of the reporter plasmids with various lengths of the lox-1 promoter were kindly provided by Dr. Jawahar L. Mehta (Department of Internal Medicine, University of Arkansas for Medical Sciences) 29. Additional lox-1 promoter luciferase reporter constructs with shorter promoter regions were created by PCR and enzyme ligation using p-2336/+36-Luc as a template (see the following for details). The PDGF-βR promoter luciferase reporter plasmid pPDGF-βR-Luc (pβ12) was a gift from Dr. Keiko Funa (Ludwig Institute for Cancer Research, Uppsala, Sweden) 30. The type I TGF-β receptor promoter luciferase reporter plasmid pTβ-RI-Luc (pES1.0) was kindly provided by Dr Michael Centrella (Yale University, New Haven, CT, USA) 31. The PPARγ cDNA expression plasmid pPPARγ, containing a full size of PPARγ cDNA, was a gift from Dr. Reed Graves (Department of Medicine, University of Chicago). The dominant negative PPARγ expression construct pdn-PPARγ was a gift from Dr. Krishna V. Chatterjee 32. The Wnt signaling luciferase reporter plasmids TOPflash and its mutant FOPflash were kindly provided by Dr. Randall T. Moon (Department of Pharmacology, School of Medicine, University of Washington) 33. TOPflash contained 8 copies of TCF/LEF binding sites. FOPflash was a counterpart control for TOPflash with site-directed mutations in the TCF/LEF binding sites 33.\nTransient transfection of semi-confluent HSCs in 6-well plates was performed using the LipofectAMINE® reagent (Invitrogen Corp.), as we previously described 23. Each treatment had triplicates in every experiment. Each experiment was repeated, at least, three times. Luciferase activity assays were conducted as we previously described 23. Transfection efficiency was determined by co-transfection of a β-galactosidase reporter, pSV-β-gal (Promega, Madison, WI). β-galactosidase assays were performed using an assay kit from Promega Corp, according to the manufacturer's instruction. Luciferase activities were expressed as relative unit after normalization with β-galactosidase activities. 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