MATERIALS AND METHODS

Cell culture
H4 neuroglioma (ATCC HTB-148) and HEK293T (ATCC CRL-11268) cells were cultured in DMEM-based media (Sigma D6429) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich F6178) and 1% penicillin–streptomycin (Corning 30-002-CI) or 1% penicillin–­streptomycin–glutamine (Corning 30-009-CI), respectively. U-2 OS cells (ATCC HTB-96) were cultured in McCoy 5A based media (Lonza 12-688F) supplemented with 10% FBS and 1% penicillin–streptomycin. Cells were lysed by resuspension and incubation in lysis buffer (200 mM NaCl,100 mM Tris-HCl, 0.5% sodium deoxycholate, 1% Triton X-100, 0.2% SDS, 660 mM phenylmethylsulfonyl fluoride, 100 μl protease inhibitor cocktail (Sigma P8215), 100 μl phosphatase inhibitor cocktail (Sigma P0044), 1250 U Benzonase nuclease) while on ice.
The following drugs were used to treat the human cell lines: calicheamicin-γ (a generous gift from Pfizer), calyculin-A (Sigma C5552), camptothecin (Sigma C9911), etoposide (Sigma E1383), doxorubicin (Cayman Chemical 15007), okadaic acid (Sigma O9381), bleomycin (Cayman Chemical 13877), DMSO (Sigma D8418), DNA-PK inhibitor (Selleckchem NU7441), sorbitol (Sigma A1876), and Adox (Sigma A7154). UV irradiation was carried out by a standard germicidal ultraviolet light with 95% of the ultraviolet radiation in the 254-nm region at 37°C. Ionizing radiation exposures were performed under 34 Gy/h in a 137Cs irradiator (GammaCell 40; J. L. Shepard and Associates) at 30°C. All drug treatments or radiation exposures were carried out for 1 h at 37°C unless otherwise specified.

Western blotting and immunoprecipitation
Cell lysates were mixed with 4X NuPAGE LDS Sample Buffer (Thermo Fisher NP0008) and run through SDS–PAGE using precast gels (Bio-Rad 456-9034). Protein was transferred onto nitrocellulose membrane (Bio-Rad 162-0146) using the Trans-Blot Turbo Transfer System (Bio-Rad 170-4150). Membranes were blocked in 6% milk (Bio-Rad 1706404XTU) in Tris-buffered saline (TBS) with 0.1% Tween-20 or Odyssey Blocking Buffer (LI-COR 927-40000). Primary and secondary antibodies were diluted in phosphate-buffered saline with 0.1% Tween-20. Secondary antibodies were conjugated to IRDye fluorochromes (LI-COR 926-32211; 925-68020), and their fluorescence was detected using the Odyssey LCx Imaging System (LI-COR). Biological replicates (n) were produced from separately cultured cells. Densitometry analysis was done using the Odyssey LI-COR Image Studio program. All data were displayed as the mean value with error bars representing 95% confidence intervals, unless otherwise stated in the figure legends, using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, CA, www.graphpad.com).
Protein G–conjugated Dynabeads (Invitrogen 10007D) and 5 µg of antibody were used to immunoprecipitate FUS from mammalian cell lysates following the manufacturer’s protocol. The following FUS antibodies were used: Bethyl A300-293A and custom-produced rabbit anti-FUS(Ser-30) phosphorylated and unphosphorylated peptides (PAN).

Immunocytochemistry and ImageJ quantification
Cells were grown on glass coverslips for 24 h prior to indicated treatments and fixation with 4% paraformaldehyde (Sigma-Aldrich P6148). The fixed cells were permeabilized in –20°C methanol and blocked in 5% normal goat serum containing 0.05% sodium azide (Life Technologies 50062Z) in preparation for immunostaining. Mouse anti-FUS (Santa Cruz 373698) and custom-produced rabbit antibodies against phosphorylated FUS (Genscript) antibodies were used as primary antibodies to probe the fixed cells. AF488 and AF586 conjugated secondary antibodies (Southern Biotech 1030-30; Life Technologies A11011) were used to detect the primary antibodies, and DAPI-containing mounting media (Invitrogen P36931) was used to stain the nuclei and mount the coverslips to glass slides. The Zeiss 700 confocal microscope was used to view and image the prepared slides. Biological replicates (n) were produced from separately cultured cells.
Quantification was performed using Fiji (Schindelin et al., 2012). For each immunofluorescence microscopy image shown, between 5 and 10 cells were quantified per biological replicate for statistical analysis. Quantification of subcellular localization was done using the Raw Integrated Density measurement. Nuclear and cytoplasmic areas were mapped under the highest maximum threshold setting and a minimum threshold setting of 10 and 1, respectively. Quantitative measurements were made without applied thresholds. Knockdown experiments were quantified using the mean gray value measurement with the same mapping techniques. All data are displayed as the mean value with error bars representing 95% confidence intervals using GraphPad Prism version 7.00 for Windows (GraphPad Software).

Phosphatase treatment of FUS protein
Immunoprecipitated FUS was treated with calf intestinal phosphatase (CIP; NEB M0290L) in a reaction buffer (100 mM NaCl, 50 mM Tris-HCl pH 8, 10 mM MgCl2, 1 mM dithiothreitol (DTT), 1 EDTA-free protease inhibitor tablet (1 tablet per 5 ml of solution; Roche 11873580001)) at 37°C for 90 min. Mock treatment contained the reaction buffer without the enzyme. The resulting reactions were separated by SDS–PAGE for Western blot analysis.

Plasmids, knockdown, and mammalian cell transfection
Wild-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.
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).

Custom peptide and antibody production and testing
Pairs of phosphorylated and nonphosphorylated peptides were synthesized corresponding to FUS residues 22–36 (GQGYSQQSSQPYGQQ; Ser-26 underlined) and residues 26–40 (SQQSSQPYGQQSYSG; Ser-30 underlined). The phosphorylated peptides were used as immunogens for antibody production in rabbits. Following immunological challenge with each phosphopeptide, antibodies specific to the phosphorylated peptides were purified: anti-FUS(pSer26) and anti-FUS(pSer30). Nonspecific antibodies that recognized both PAN were also isolated. Peptide synthesis and antibody production were performed by Genscript (Piscataway, NJ).
Phosphorylated and nonphosphorylated peptides were blotted onto nitrocellulose and probed with anti-FUS(pSer26) and anti-FUS(pSer30) antibodies following normal Western blotting protocols. Knockdown of FUS from H4 cells using Lipofectamine RNAiMAX (ThermoFisher 13778030) and Silencer Select siRNA specific to FUS (ThermoFisher 4392420 and 4390843) was done following manufacturer’s protocol to confirm antibody specificity in cells.

Mass spectrometry
H4 cells were treated with 50 nM calicheamicin or 30 µM camptothecin for 1 h at 37°C and then lysed in nondenaturing lysis buffer (described above). FUS was immunoprecipitated (procedure described above) from the samples using protein G–conjugated Dynabeads (Invitrogen) and the custom produced polyclonal anti-FUS(Ser-30) PAN antibody (Genscript), which recognizes both phosphorylated and unphosphorylated FUS. Samples were enriched for the phosphorylated population of FUS using a titanium dioxide column (ThermoFisher Scientific). The protein was eluted, cleaved with trypsin and then chymotrypsin to isolate the N-terminal prionlike domain of FUS. Mass spectrometry analysis was then performed at the John Hopkins University Mass Spectrometry and Proteomic Facility. This protocol was previously described by Monahan and colleagues (Monahan et al. 2017). PEAKS Studio Software was used to view and analyze the mass spectrometry data.