PMC:2586094 / 4593-7785 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 Materials and methods\n\n2.1 Materials\nCell lines (mouse macrophage J774.2, human monocytic U937) were from the European Collection of Animal Cell Cultures. Antibodies were from Santa Cruz Biotechnology (casein kinase 2 (CK2)-α and α′) and Upstate Biotechnology (Sp1 and Sp3). The protein kinase B (PKB) activity assay kit was from Cell Signalling Technology. Recombinant IFN-γ and tumour necrosis factor-α (TNF-α) were from Peprotech, and recombinant Sp1 and purified CK2 enzyme were from Promega.\n\n2.2 Cell culture\nCells were maintained in Dulbecco's modified Eagle's medium (J744.2) or RPMI 1640 (U937) supplemented with heat-inactivated (56 °C, 30 min) foetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. The cultures were maintained at 37 °C in a humidified atmosphere containing 5% (v/v) CO2 in air. Treatment of J774.2 macrophages with IFN-γ (1000 U/ml) or the different pharmacological inhibitors was performed as previously described [9–14]. U937 cells were used for all transfection assays where differentiation was initiated using 1 µM phorbol-12-myristate-13-acetate (PMA) and monitored by morphological analysis [9,15].\n\n2.3 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.\n\n2.4 Western blot analysis\nWhole cell extracts were prepared in buffers containing phosphatase and protease inhibitors and used for SDS-PAGE and western blot analysis as previously described [9–13,15].\n\n2.5 Immunoprecipitations and CK2 activity assays\nWhole cell extracts (150–300 µg) were prepared using buffers containing phosphatase and protease inhibitors and subjected to immunoprecipitation and in vitro kinase assays using the β-casein substrate as previously described [10]. For CK2 assays with Sp1, 0.5 µg of recombinant protein was used as a substrate instead of β-casein. For co-immunoprecipitation assays, the immunoprecipitated proteins were eluted using 0.1 M glycine (pH 2.5) and subjected to SDS-PAGE and western blot analysis.\n\n2.6 Electrophoretic mobility shift assays (EMSA)\nEMSA were carried out using whole cell extracts and radiolabelled oligonucleotides against the Sp1 binding sites in the LPL gene as previously described [9,15].\n","divisions":[{"label":"Title","span":{"begin":3,"end":24}},{"label":"Section","span":{"begin":26,"end":500}},{"label":"Title","span":{"begin":31,"end":40}},{"label":"Section","span":{"begin":502,"end":1150}},{"label":"Title","span":{"begin":507,"end":519}},{"label":"Section","span":{"begin":1152,"end":2233}},{"label":"Title","span":{"begin":1157,"end":1204}},{"label":"Section","span":{"begin":2235,"end":2436}},{"label":"Title","span":{"begin":2240,"end":2261}},{"label":"Section","span":{"begin":2438,"end":2979}},{"label":"Title","span":{"begin":2443,"end":2487}},{"label":"Section","span":{"begin":2981,"end":3191}},{"label":"Title","span":{"begin":2986,"end":3030}}],"tracks":[{"project":"2_test","denotations":[{"id":"18793716-11796707-20139048","span":{"begin":964,"end":966},"obj":"11796707"},{"id":"18793716-12609974-20139048","span":{"begin":964,"end":966},"obj":"12609974"},{"id":"18793716-17255531-20139048","span":{"begin":964,"end":966},"obj":"17255531"},{"id":"18793716-8891433-20139048","span":{"begin":964,"end":966},"obj":"8891433"},{"id":"18793716-9505144-20139048","span":{"begin":964,"end":966},"obj":"9505144"},{"id":"18793716-10419646-20139048","span":{"begin":964,"end":966},"obj":"10419646"},{"id":"18793716-11796707-20139049","span":{"begin":1146,"end":1148},"obj":"11796707"},{"id":"18793716-15755745-20139049","span":{"begin":1146,"end":1148},"obj":"15755745"},{"id":"18793716-11796707-20139050","span":{"begin":1434,"end":1436},"obj":"11796707"},{"id":"18793716-15755745-20139050","span":{"begin":1434,"end":1436},"obj":"15755745"},{"id":"18793716-11796707-20139051","span":{"begin":2432,"end":2434},"obj":"11796707"},{"id":"18793716-12609974-20139051","span":{"begin":2432,"end":2434},"obj":"12609974"},{"id":"18793716-17255531-20139051","span":{"begin":2432,"end":2434},"obj":"17255531"},{"id":"18793716-8891433-20139051","span":{"begin":2432,"end":2434},"obj":"8891433"},{"id":"18793716-9505144-20139051","span":{"begin":2432,"end":2434},"obj":"9505144"},{"id":"18793716-15755745-20139051","span":{"begin":2432,"end":2434},"obj":"15755745"},{"id":"18793716-12609974-20139052","span":{"begin":2714,"end":2716},"obj":"12609974"},{"id":"18793716-11796707-20139053","span":{"begin":3187,"end":3189},"obj":"11796707"},{"id":"18793716-15755745-20139053","span":{"begin":3187,"end":3189},"obj":"15755745"}],"attributes":[{"subj":"18793716-11796707-20139048","pred":"source","obj":"2_test"},{"subj":"18793716-12609974-20139048","pred":"source","obj":"2_test"},{"subj":"18793716-17255531-20139048","pred":"source","obj":"2_test"},{"subj":"18793716-8891433-20139048","pred":"source","obj":"2_test"},{"subj":"18793716-9505144-20139048","pred":"source","obj":"2_test"},{"subj":"18793716-10419646-20139048","pred":"source","obj":"2_test"},{"subj":"18793716-11796707-20139049","pred":"source","obj":"2_test"},{"subj":"18793716-15755745-20139049","pred":"source","obj":"2_test"},{"subj":"18793716-11796707-20139050","pred":"source","obj":"2_test"},{"subj":"18793716-15755745-20139050","pred":"source","obj":"2_test"},{"subj":"18793716-11796707-20139051","pred":"source","obj":"2_test"},{"subj":"18793716-12609974-20139051","pred":"source","obj":"2_test"},{"subj":"18793716-17255531-20139051","pred":"source","obj":"2_test"},{"subj":"18793716-8891433-20139051","pred":"source","obj":"2_test"},{"subj":"18793716-9505144-20139051","pred":"source","obj":"2_test"},{"subj":"18793716-15755745-20139051","pred":"source","obj":"2_test"},{"subj":"18793716-12609974-20139052","pred":"source","obj":"2_test"},{"subj":"18793716-11796707-20139053","pred":"source","obj":"2_test"},{"subj":"18793716-15755745-20139053","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93cbec","default":true}]}]}}