Phosphomimetic FUS Variants in Cellular Assays

Wild-type FUS and its phosphomimetic variants were subcloned from pET vectors previously constructed (Monahan et al., 2017 (link)) 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.
Phosphomimetic 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).

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Rhoads S.N., Monahan Z.T., Yee D.S., Leung A.Y., Newcombe C.G., O’Meally R.N., Cole R.N, & Shewmaker F.P. (2018). The prionlike domain of FUS is multiphosphorylated following DNA damage without altering nuclear localization. Molecular Biology of the Cell, 29(15), 1786-1797.

Publication 2018

mEGFP-C1 OptiMEM Lipofectamine 2000

Cell Cell lines Gene Lipofectamine 2000 Plasmid Transfection Vectors

Corresponding Organization : Uniformed Services University of the Health Sciences

Other organizations : Johns Hopkins University

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Variable analysis

independent variables

Wild-type FUS
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)

dependent variables

Transfection efficiency
Protein expression levels

control variables

DNA-to-Lipofectamine 2000 ratio (0.5 µg: 2.5 µl)
Transfection mixture dilution (1:4 into complete cell media)
Incubation time (8-10 hours)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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