PMC:2586094 / 5745-6826 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2586094","sourcedb":"PMC","sourceid":"2586094","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2586094","text":"2.3 Transient transfection and reporter gene assays\nTransfection of U937 cells with an LPL promoter-luciferase DNA construct (− 31 to + 187) and CMV-β-galactosidase (internal control for transfection efficiency) was carried out using SuperFect™ (Qiagen) as previously described [9,15]. In experiments involving the use of dominant negative (DN) constructs, the cells were initially transfected with the DN expression plasmid or control vector and incubated for 8 h before transfection of the cells with the LPL promoter-luciferase and CMV-β galactosidase plasmids. The DN plasmids used were pcDNA3 HA-PKB AAA from Dr. B. Hemmings (Friedrich Miescher Institute for Biomedical Research, Basel) and pSG-CK2α-K68A from Drs. E. M. Chambaz and C. Cochet (INSERM, Grenoble). DN PKB specifies for an inactive form of PKBα with a mutation to alanine of lysine 179 in the ATP-binding site and threonine 308 and serine 473 that must be phosphorylated in the active kinase. DN CK2 codes for a kinase inactive mutant with a lysine to alanine change at residue 68 within the ATP-binding domain.","divisions":[{"label":"Title","span":{"begin":5,"end":52}}],"tracks":[{"project":"2_test","denotations":[{"id":"18793716-11796707-20139050","span":{"begin":282,"end":284},"obj":"11796707"},{"id":"18793716-15755745-20139050","span":{"begin":282,"end":284},"obj":"15755745"}],"attributes":[{"subj":"18793716-11796707-20139050","pred":"source","obj":"2_test"},{"subj":"18793716-15755745-20139050","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ebec93","default":true}]}]}}