PMC:3583298 / 34720-35750 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3583298","sourcedb":"PMC","sourceid":"3583298","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3583298","text":"Protein preparation and immunoblotting\nProtein extracts were prepared as previously described (Kenney and Rowitch, 2000). Protein content was determined by using the Bio-Rad protein assay. 50-75 μg of each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8-10% polyacrylamide gels and then transferred in 20% methanol buffer at 4°C to Immobilon polyvinylidene difluoride (Millipore) membranes. Immunoblotting was carried out according to standard methods. Antibodies used for western blotting were: YAP1 (Abcam), phospho-Akt (Ser473), phospho-Cdk1(Tyr15), phospho-Chk2 (Thr68), total Akt and cleaved caspase 3 (Cell Signaling), phospho-ATM (Ser1981) (Rockland), Chk2 (Millipore), IGF2 and β-tubulin (Sigma), VEGF, Cyclin D2 and Cyclin B1 (Santa Cruz). Donkey anti-mouse HRP-linked secondary was from Jackson Research Laboratories and goat anti-rabbit from Thermo Scientific. Peroxidase activity was detected using Amershams’s ECL reagents and exposing membranes to Kodak Biomax film.","divisions":[{"label":"title","span":{"begin":0,"end":38}}],"tracks":[{"project":"2_test","denotations":[{"id":"21874045-11074003-79138736","span":{"begin":115,"end":119},"obj":"11074003"},{"id":"21874045-11074003-79138736","span":{"begin":115,"end":119},"obj":"11074003"}],"attributes":[{"subj":"21874045-11074003-79138736","pred":"source","obj":"2_test"},{"subj":"21874045-11074003-79138736","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#96ec93","default":true}]}]}}