PMC:6085830 / 41181-42145 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6085830","sourcedb":"PMC","sourceid":"6085830","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6085830","text":"Plasmids, knockdown, and mammalian cell transfection\nWild-type FUS and its phosphomimetic variants were subcloned from pET vectors previously constructed (Monahan et al., 2017) into both mEGFP-C1 (Addgene 54759) and pIRES2-EGFP-SHP2DA (Add­gene 12286) using BamHI/XhoI restriction sites. The phosphomimetic substitution in each construct are listed below. Plasmid DNA was transfected into cell lines using OptiMEM (Life Technologies 31985-070) transfection medium and Lipofectamine 2000 (Thermo Fisher 11668027) at a ratio of 0.5 µg: 2.5 µl (DNA: Lipofectamine 2000). Transfection mixture was diluted 1:4 into complete cell media and incubated at 37°C for 8–10 h.\nPhosphomimetic substitutions in FUS are diagrammed in Supplemental Figure S8 and are as follows: FUS-1E (T19E); FUS-3E (S26E, S30E, T68E); FUS-4E (T68E, S84E, S87E, S117E); FUS-6E (S26E, S30E, T68E, S84E, S87E, S117E); FUS-12E (T7E, T11E, T19E, S26E, S30E, S42E, S61E, T68E, S84E, S87E, S117E S131E).","divisions":[{"label":"Title","span":{"begin":0,"end":52}}],"tracks":[{"project":"2_test","denotations":[{"id":"29897835-28790177-70504571","span":{"begin":171,"end":175},"obj":"28790177"}],"attributes":[{"subj":"29897835-28790177-70504571","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93b6","default":true}]}]}}